Compositions and methods for editing beta-globin for treatment of hemaglobinopathies

ABSTRACT

The disclosure features methods of correcting a mutation in the human beta-globin (HBB) gene in a cell or population of cells. The disclosure also features methods of increasing repair of a DNA double stranded break (DSB) in an HBB gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.

RELATED APPLICATIONS

This application is a U.S. National Phase Application, filed under 35U.S.C. § 371, of International Application No. PCT/US2020/038203, filedon Jun. 17, 2020, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/862,539, filed Jun. 17, 2019, which are herebyincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created onAug. 16, 2022, is named VTEX_002_N01US_SubSeqList_ST25.txt and is136,408 bytes in size.

BACKGROUND

Hemoglobin (Hb) carries oxygen from the lungs to tissues in erythrocytesor red blood cells (RBCs). During prenatal development and until shortlyafter birth, hemoglobin is present in the form of fetal hemoglobin(HbF), a tetrameric protein composed of two alpha (a)-globin chains andtwo gamma (γ)-globin chains. HbF is largely replaced by adult hemoglobin(HbA), a tetrameric protein in which the γ-globin chains of HbF arereplaced with beta (β)-globin chains, through a process known as globinswitching. HbF is more efficient than HbA at carrying oxygen. Theaverage adult makes less than 1% HbF out of total hemoglobin. Theα-hemoglobin gene is located on chromosome 16, while the β-hemoglobingene (HBB), A gamma (γ^(A))-globin chain (HBG1, also known as gammaglobin A), and G gamma (γ{circumflex over ( )}-globin chain (HBG2, alsoknown as gamma globin G) are located on chromosome 11 within the globingene cluster (i.e., globin locus).

Mutations in HBB can cause hemoglobin disorders (i.e.,hemoglobinopathies) including sickle cell disease (SCD) andbeta-thalassemia (β-Thal). Approximately 93,000 people in the UnitedStates are diagnosed with a hemoglobinopathy. Worldwide, 300,000children are born with hemoglobinopathies every year (Angastiniotis &Modell, Ann N Y Acad Sci, 850:251-269 (1998)). Because these conditionsare associated with HBB mutations, their symptoms typically do notmanifest until after globin switching from HbF to HbA.

SCD is the most common inherited hematologic disease in the UnitedStates, affecting approximately 80,000 people (Brousseau, Am J Hematol85(1):77-78 (2010)). SCD is most common in people of African ancestry,for whom the prevalence of SCD is 1 in 500. In Africa, the prevalence ofSCD is 15 million (Aliyu et al. Am J Hematol, 83:63-70 (2008)). SCD isalso more common in people of Indian, Saudi Arabian and Mediterraneandescent.

SCD is caused by a single homozygous mutation in the HBB gene, c.20A>T(HbS mutation). The sickle mutation is a point mutation (GAG-GTG) on HBBthat results in substitution of valine for glutamic acid at amino acidposition 6 in exon 1 (E6V) in the protein. The mutation is also referredto as an E7V mutation because it occurs at the 7^(th) position in thegene coding exon, where the first amino acid is methionine. The valineat position 6 of the β-hemoglobin chain is hydrophobic and causes achange in conformation of the β-globin protein when it is not bound tooxygen. This change of conformation causes HbS proteins to polymerize inthe absence of oxygen, leading to deformation (i.e., sickling) of RBCs.SCD is inherited in an autosomal recessive manner, so that only patientswith two HbS alleles have the disease. Heterozygous subjects have sicklecell trait, and may suffer from anemia and/or painful crises if they areseverely dehydrated or oxygen deprived.

Delivery of a corrected HBB gene via gene therapy is currently beinginvestigated in clinical trials. However, the long-term efficacy andsafety of this approach is unknown. Transplantation with hematopoieticstem cells from an HLA-matched allogeneic stem cell donor has beendemonstrated to cure SCD, but this procedure involves risks includingthe possibility of graft vs. host disease after transplantation. Inaddition, matched allogeneic donors often cannot be identified. Thus,there is a need for improved methods of managing these and otherhemoglobinopathies.

SUMMARY OF DISCLOSURE

In some aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells the methodcomprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease, whereinthe S. pyogenes Cas9 endonuclease is a high fidelity Cas9;

(b) a single guide RNA (sgRNA) targeting a target site in an HBB gene,the sgRNA comprising a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 15; and

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene;

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene.

In other aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells the methodcomprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease, whereinthe S. pyogenes Cas9 endonuclease is a high fidelity Cas9;

(b) a single guide RNA (sgRNA) targeting a target site in an HBB gene,the sgRNA recognizes a target sequence consisting of SEQ ID NO: 20; and

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene;

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene.

In any of the foregoing or related aspects, cleavage of one or morepredicted off-target sites in the cell or population of cells is reducedrelative to a cell or population of cells contacted with a wild-type S.pyogenes Cas9. In some aspects, cleavage of one or more predictedoff-target sites is reduced by at least about 50%.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(ii) corrects an E6V mutation in the HBB gene and is homologous witha region of the HBB gene encoding the E6V mutation. In some aspects, thenucleotide sequence of (c)(ii) comprises the sequence of SEQ ID NO: 102.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(i) is homologous with a region located upstream of the E6V mutationin the HBB gene and the nucleotide sequence of (c)(iii) is homologouswith a region located downstream of the E6V mutation.

In some aspects, the disclosure provides a method for correcting an E6Vmutation in human beta-globin (HBB) in a cell or population of cells,the method comprising contacting the cell or population of cellscomprising an HBB gene encoding the E6V mutation with:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene;and

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of HBB gene, thereby correcting the E6V mutation in the HBBgene in the cell or population of cells.

In any of the foregoing or related aspects, the DSB occurs 10-50nucleotides upstream or downstream of the region of the HBB geneencoding the E6V mutation. In some aspects, the DSB occurs within exon 1of the HBB gene.

In any of the foregoing or related aspects, the sgRNA comprises a spacersequence corresponding to a target sequence consisting of SEQ ID NO: 15.In other aspects, the sgRNA recognizes a target sequence consisting ofSEQ ID NO: 15. In other aspects, the sgRNA recognizes a target sequenceconsisting of SEQ ID NO: 20.

In any of the foregoing or related aspects, the DNA endonuclease is aCas9 endonuclease. In some aspects, the Cas9 endonuclease is a S.pyogenes Cas9 endonuclease.

In some aspects, the disclosure provides a method for correcting an E6Vmutation in human beta-globin (HBB) in a cell or population of cells,the method comprising contacting the cell or population of cellscomprising an HBB gene encoding the E6V mutation with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene,the sgRNA comprising a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 15; and

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene, thereby correcting the E6V mutation in the HBBgene in the cell or population of cells.

In some aspects, the disclosure provides a method for correcting an E6Vmutation in human beta-globin (HBB) in a cell or population of cells,the method comprising contacting the cell or population of cellscomprising an HBB gene encoding the E6V mutation with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene,the sgRNA recognizes a target sequence consisting of SEQ ID NO: 20; and

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene, thereby correcting the E6V mutation in the HBBgene in the cell or population of cells.

In any of the foregoing or related aspects, the method further comprisescontacting the cell with a 53BP1 inhibitor. In some aspects, the methodfurther comprises contacting the cell with an inhibitor of DNA-PK. Inother aspects, the method comprises contacting the cell with a 53BP1inhibitor and an inhibitor of DNA-PK. In some aspects, the 53BP1inhibitor and/or the inhibitor of DNA-PK increases HDR of the DSB,relative to HDR in a cell or population of cells without the 53BP1inhibitor and/or inhibitor of DNA-PK.

In any of the foregoing or related aspects, the S. pyogenes Cas9endonuclease is a high fidelity S. pyogenes Cas9 endonuclease. In someaspects, cleavage of one or more predicted off-target sites is reducedby at least about 50% relative to a cell or population of cellscontacted with a wild-type S. pyogenes Cas9.

In some aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in an HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene; and

(d) a 53BP1 inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the HDR of the DSB isincreased relative to HDR in a cell or population of cells without the53BP1 inhibitor.

In other aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in an HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene; and

(d) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the HDR of the DSB isincreased relative to HDR in a cell or population of cells without theDNA-PK inhibitor.

In other aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in an HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene;

(d) a 53BP1 inhibitor; and

(e) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the HDR of the DSB with theexchange of the nucleic acid into the HBB gene in the cell or populationof cells is increased relative to HDR in a cell or population of cellswithout the 53BP1 inhibitor and DNA-PK inhibitor.

In any of the foregoing or related aspects, the DSB occurs 10-50nucleotides upstream or downstream of a region of the HBB gene encodingan E6V mutation. In some aspects, the DSB occurs within exon 1 of theHBB gene.

In any of the foregoing or related aspects, the sgRNA comprises a spacersequence corresponding to a target sequence consisting of SEQ ID NO: 15.In other aspects, the sgRNA recognizes a target sequence consisting ofSEQ ID NO: 15. In some aspects, the sgRNA recognizes a target sequenceconsisting of SEQ ID NO: 20.

In some aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene,the sgRNA comprising a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 15;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene; and

(d) a 53BP1 inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the HDR of the DSB isincreased relative to HDR in a cell or population of cells without the53BP1 inhibitor.

In other aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene,the sgRNA comprising a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 15;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene; and

(d) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the DSB is increased relativeto HDR in a cell or population of cells without the DNA-PK inhibitor.

In other aspects, the disclosure provides a method for homology directedrepair (HDR) of a double-strand break (DSB) in a target region in ahuman beta-globin (HBB) gene in a cell or population of cells, themethod comprising contacting the cell or population of cells with:

(a) a S. pyogenes Cas9 endonuclease, an mRNA encoding the S. pyogenesCas9 endonuclease, or a recombinant expression vector comprising anucleotide sequence encoding the S. pyogenes Cas9 endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in the HBB gene,the sgRNA comprising a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 15;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the target region in the HBB gene, (ii) anucleotide sequence homologous with a region of the HBB gene comprisingthe target region, and (iii) a nucleotide sequence homologous with aregion located downstream of the target region in the HBB gene;

(d) a 53BP1 inhibitor; and

(e) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, the HDR of the DSB isincreased relative to HDR in a cell or population of cells without the53BP1 inhibitor and DNA-PK inhibitor.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(ii) corrects an E6V mutation in the HBB gene and is homologous witha region of the HBB gene encoding an E6V mutation. In some aspects, thenucleotide sequence of (c)(ii) comprises the sequence of SEQ ID NO: 102.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(i) is homologous with a region upstream of the region encoding anE6V mutation in the HBB gene and the nucleotide sequence of (c)(iii) ishomologous to a region downstream of the E6V mutation.

In some aspects, the disclosure provides a method for correcting an E6Vmutation in human beta-globin (HBB) by homology directed repair (HDR) ina cell or population of cells, the method comprising contacting the cellor population of cells comprising an HBB gene encoding the E6V mutationwith:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in a HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene; and

(d) a 53BP1 inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, correction of the E6Vmutation is increased relative to a cell or population of cells withoutthe 53BP1 inhibitor.

In other aspects, the disclosure provides a method for correcting an E6Vmutation in human beta-globin (HBB) by homology directed repair (HDR) ina cell or population of cells, the method comprising contacting the cellor population of cells comprising an HBB gene encoding the E6V mutationwith:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in a HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene; and

(d) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, correction of the E6Vmutation is increased relative to a cell or population of cells withoutthe DNA-PK inhibitor.

In yet other aspects, the disclosure provides a method for correcting anE6V mutation in human beta-globin (HBB) by homology directed repair(HDR) in a cell or population of cells, the method comprising contactingthe cell or population of cells comprising an HBB gene encoding the E6Vmutation with:

(a) a DNA endonuclease, an mRNA encoding the DNA endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe DNA endonuclease;

(b) a single guide RNA (sgRNA) targeting a target site in a HBB gene;

(c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene;

(d) a 53BP1 inhibitor; and

(e) a DNA-PK inhibitor,

wherein a double-strand break (DSB) occurs at the target site in the HBBgene and the nucleic acid is exchanged with a homologous nucleotidesequence of the HBB gene. In some aspects, correction of the E6Vmutation is increased relative to a cell or population of cells withoutthe 53BP1 inhibitor and DNA-PK inhibitor.

In any of the foregoing or related aspects, the DSB occurs 10-50nucleotides upstream or downstream of a region of the HBB gene encodingthe E6V mutation. In some aspects, the DSB occurs within exon 1 of theHBB gene.

In any of the foregoing or related aspects, the sgRNA comprises a spacersequence corresponding to a target sequence consisting of SEQ ID NO: 15.In other aspects, the sgRNA recognizes a target sequence consisting ofSEQ ID NO: 15. In other aspects, the sgRNA recognizes a target sequenceconsisting of SEQ ID NO: 20.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(i) is homologous with a region comprising the promoter of the HBBgene and/or upstream sequences of the coding region of the HBB gene.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(iii) is homologous with a region comprising a portion of exon 1,intron 1-2, exon 2, and a portion of intron 2-3, inclusive, and,optionally all or a portion of exon 3, of the HBB gene. In some aspects,the nucleotide sequence of (c)(iii) spans the target site.

In any of the foregoing or related aspects, the DNA endonuclease is aCas9 endonuclease. In some aspects, the Cas9 endonuclease is a S.pyogenes Cas9 endonuclease. In some aspects, the S. pyogenes Cas9endonuclease is a high fidelity S. pyogenes Cas9 endonuclease. In someaspects, cleavage of one or more predicted off-target sites is reducedby at least about 50% relative to a wild-type S. pyogenes Cas9endonuclease. In any of the foregoing or related aspects, the highfidelity Cas9 endonuclease comprises a R691A mutation. In some aspects,the high fidelity Cas9 endonuclease comprises at least one NLS. In someaspects, the at least one NLS is an sv40 NLS.

In any of the foregoing or related aspects, the 53BP1 inhibitor and/orthe inhibitor of DNA-PK increases HDR frequency in the cell populationby at least 50% relative to a cell population without the 53BP1inhibitor and/or the inhibitor of DNA-PK. In some aspects, the 53BP1inhibitor and/or the inhibitor of DNA-PK decreases indel frequency by1-2 fold, 1.1-2 fold, or 2-10 fold in the cell population.

In any of the foregoing or related aspects, the 53BP1 inhibitor is a53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB inthe cell. In some aspects, the 53BP1 binding polypeptide comprises anamino acid sequence selected from a group consisting of: SEQ ID NOs: 70,74, 77, 80, 83 and 86. In some aspects, the 53BP1 inhibitor comprises anucleic acid comprising a nucleotide sequence encoding a 53BP1 bindingpolypeptide that inhibits 53BP1 recruitment to the DSB site in the cell.In some aspects, the nucleic acid comprises a nucleotide sequenceselected from a group consisting of: SEQ ID NOs: 69, 73, 76, 79, 82, 85and 88. In some aspects, the nucleic acid comprises a vector comprisinga nucleotide sequence encoding the 53BP1 binding polypeptide. In someaspects, the vector comprises a nucleotide sequence selected from agroup consisting of: SEQ ID NOs: 68, 72, 75, 78, 81, 84 and 87. In otheraspects, the 53BP1 inhibitor comprises a small interfering ribonucleicacid (siRNA) targeting 53BP1.

In any of the foregoing or related aspects, the inhibitor of DNA-PKtargets the catalytic subunit of DNA-PK (DNA-PKcs). In some aspects, theinhibitor of DNA-PK is Nu7441. In some aspects, the inhibitor of DNA-PKis Compound 984 or Compound 296.

In any of the foregoing or related aspects, the nucleotide sequence of(c)(i) is about 0.2 kb to about 3 kb in length. In any of the foregoingor related aspects, the nucleotide sequence of (c)(iii) is about 0.2 kbto about 3 kb in length. In any of the foregoing or related aspects, thenucleotide sequence of (c)(i) and/or the nucleotide sequence of (c)(iii)is about 0.2 kb-1 kb, about 1 kb-1.5 kb, 1.5 kb-2 kb, 2 kb-2.2 kb or 2.0kb-2.3 kb in length. In any of the foregoing or related aspects, thenucleotide sequence of (c)(i) and/or the nucleotide sequence of (c)(iii)is about 2.2 kb each.

In any of the foregoing or related aspects, the recombinant vectorcomprises SEQ ID NO: 98. In some aspects, the recombinant vector is anAAV vector. In some aspects, the AAV vector is about 2.5 kb-4.6 kb inlength. In some aspects, the AAV vector comprises AAV6. In some aspects,the AAV vector comprises 5′ and 3′ inverted terminal repeats (ITRs)derived from AAV2. In some aspects, the 5′ ITR comprises SEQ ID NO: 106and the 3′ ITR comprises SEQ ID NO: 107. In some aspects, the AAV vectorcomprises SEQ ID NO: 105.

In any of the foregoing or related aspects, the cell or population ofcells is a hematopoietic stem or progenitor cell (HSPC). In someaspects, the cell or population of cells is a long-term HSPC (LT-HSPC).In some aspects, the cell is a CD34 expressing cell.

In any of the foregoing or related aspects, the cell or population ofcells is isolated from a tissue sample obtained from a human donor. Insome aspects, the tissue sample is a peripheral blood sample. In someaspects, the human donor has a sickle cell disease.

In some aspects, the disclosure provides a cell or population of cellsgenerated by any of the methods described herein

In some aspects, the disclosure provides a method for treating a patienthaving a disease or disorder, comprising administering the cell orpopulation of cells described herein, thereby treating the disease ordisorder. In some aspects, the disease or disorder is sickle cellanemia.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1D include bar graphs showing efficiency of HDR repair inHEK293 T cells using single-stranded oligodeoxynucleotide (ssODN) donorDNA that converts a gene in the AAVS1 locus encoding a blue fluorescentprotein (BFP) to a gene encoding green fluorescent protein (GFP). FIG.1A shows HDR efficiency in the presence of Nu7441 (e.g., an inhibitor ofDNA-PKcs), SCR7 (e.g., an inhibitor of DNA Ligase IV), and RS1 (e.g., anagonist of Rad51). FIG. 1B shows HDR efficiency in the presence ofNu7441 or Veliparib (e.g., an inhibitor of PARP) with varied doses ofinhibitor. FIG. 1C shows HDR efficiency in the presence of Nu7441 orL755,507 (e.g., an inhibitor of β3-adrenergic receptor) using twodifferent ssODN templates with varied doses of inhibitor. FIG. 1D showsHDR efficiency in the presence of the i53 polypeptide inhibitor of 53BP1at varied doses using two different ssODN donors.

FIGS. 2A-2D include bar graphs showing editing in HEK293 T cellsfollowing electroporation with Cas9/sgRNA RNP using single-strandedoligodeoxynucleotide (ssODN) donor DNA that converts a gene in the AAVS1locus encoding GFP to a gene encoding BFP in the presence of Nu7441,SCR7 or RS1. FIGS. 2A-2B show the efficiency of HDR repair to convertGFP to BFP. FIGS. 2C-2D show indel formation in the AAVS1 locus.

FIG. 3 includes a bar graph showing efficiency of gene insertion intothe GSD1a locus in HEK293 T cells using either ssODNs as homology donorsthat facilitate HDR or dsDNA donors that facilitate NHEJ repair. Repairefficiency was evaluated in the presence of Nu7441, SCR7, or RS-1 usingtwo different ssODN donor templates and two different dsDNA donortemplates.

FIGS. 4A-4B include bar graphs showing mutations at the site of a DSBinduced by Cas9/gRNA in the CFTR locus in HEK293 T cells resulting fromDSB repair in the presence of a donor ssODN only (FIG. 4A) or donorssODN and the DNA-PK inhibitor Nu7441 (FIG. 4B).

FIG. 5 include a bar graphs showing mutations at the site of DSB inducedby Cas9/gRNA in the CFTR locus in HEK293 T cells resulting from DSBrepair in the presence of donor ssODN H3-95-30 (SEQ ID NO: 41) or donorssODN N1-95-30 (SEQ ID NO: 42) with treatment of Nu7441. Control cellsare electroporated in the absence of gene-editing components or Nu7441(“mock+DMSO”).

FIGS. 6A-6C include bar graphs showing HDR editing efficiency forinsertion of donor DNA encoding GFP into the hemoglobin subunit beta(HBB) locus of CD34-expressing long-term repopulating hematopoietic stemcells (LT-HSPCs) using AAV-mediated delivery of donor DNA encoding GFP.FIG. 6A shows HDR efficiency in the presence of different doses of mRNAencoding i53 (e.g., inhibitor of 53BP1) relative to negative controlsthat include mock electroporation (EP), AAV donor DNA alone or RNP-only(i.e., no AAV donor DNA). FIG. 6B shows HDR efficiency in the presenceof different doses of mRNA encoding i53, Cyren1 (e.g., inhibitor ofKu70/80), or Cyren2 (e.g., inhibitor of Ku70/80) relative to negativecontrols that include AAV alone (i.e., no RNP). FIG. 6C shows HDRefficiency in the presence of varied doses of Nu7441 relative to aDMSO-only control, mRNA encoding i53, or a control mRNA (DM) (i.e.,absence of a modulator of DNA repair).

FIG. 7 includes a dot plot showing HDR editing efficiency with treatmentof i53 for insertion of donor DNA encoding GFP delivered by AAV into theAAVS1 locus of hTERT RPE-1 cells.

FIG. 8 includes a schematic showing editing of the HBB locus using ahomology DNA donor to introduce a sickle cell correction mutation by HDRrepair of a DNA DSB formed by Cas9/gRNA complex.

FIG. 9 includes a schematic showing the sequence near the site ofCas9/gRNA gene editing within the HBB locus. Included is the sequencefor a wild type gene and for a sickle cell mutant gene. The sequencetargeted by the gRNA is highlighted, as well as the sequence of thedonor DNA that includes the sickle cell mutation. Silent mutationsencoded by the donor DNA are annotated.

FIG. 10 includes a bar graph showing HDR editing efficiency forinsertion of donor DNA encoding a sickle cell mutation into the HBBlocus of CD34-expressing HSPCs using AAV-mediated delivery of donor DNA.Shown is a comparison of HDR efficiency in the presence of i53 relativeto RNP+AAV-only, AAV-only, or RNP-only.

FIG. 11 includes a bar graph showing NHEJ editing efficiency within theHBB locus in CD34-expressing LT-HSPCs following electroporation withgRNA/Cas9 RNP and transfection with a homology DNA donor delivered byAAV. Treatment with i53 is compared to RNP+AAV-only, AAV-only, RNP-only,and mock electroporation (e.g., no RNP or AAV).

FIGS. 12A-12B include bar graphs showing HDR editing efficiency forinsertion of donor DNA encoding a sickle cell mutation into the HBBlocus of CD34-expressing LT-HSPCs using AAV-mediated delivery of donorDNA. FIG. 12A includes a bar graph showing HDR editing efficiency inCD34-expressing LT-HSPCs isolated following mobilization with acombination of Mozobil and GCSF. FIG. 12B includes a bar graph showingHDR editing efficiency in LT-HSPCs isolated following mobilization withMozobil alone.

FIG. 13 includes a bar graph showing growth of CD34-expressing LT-HSPCsduring ex vivo culture following gene editing with Cas9/gRNA RNP andAAV, either with or without treatment of i53.

FIG. 14 includes a schematic showing a schedule for administration ofgene-edited CD34-expressing LT-HSPCs into irradiated mice and subsequentanalysis of mouse tissues for engraftment and HDR editing efficiency.

FIG. 15 includes scatter plots showing a flow cytometry gating strategyfor quantification and lineage analysis of mouse tissue samples forcells derived from engrafted human LT-HSPCs.

FIG. 16 includes a bar graph showing % chimerism of human cells derivedfrom engrafted LT-HSPCs in mouse bone marrow samples isolated at 16weeks post-engraftment. CD34-expressing LT-HSPCs were administered tomice according to FIG. 14

FIG. 17 includes a bar graph showing % chimerism of human cells derivedfrom engrafted LT-HSPCs in mouse blood samples isolated at 8 and 16weeks post-engraftment. CD34-expressing LT-HSPCs were administered tomice according to FIG. 14

FIG. 18A-18B include bar graphs showing lineage distribution in mousebone marrow isolated at 16 weeks post-engraftment of LT-HSPCs. Shown isthe percentage of human CD45-expressing cells that are B cells, T cells,myeloid cells, or CD34-expressing hematopoietic stem/progenitor cells(HSPCs). CD34-expressing LT-HSPCs were administered to mice according toFIG. 14

FIG. 19 includes a dot plot showing HDR editing efficiency for insertionof donor DNA encoding a sickle cell mutation into the HBB locus,measured in mouse bone marrow isolated at 16 weeks post-engraftment ofLT-HSPCs.

FIG. 20 includes a dot plot showing indel frequency in mouse bone marrowisolated at 16 weeks post-engraftment of LT-HSPCs relative to the indelfrequency of LT-HSPCs prior to engraftment (e.g., input indels).

FIG. 21 includes a schematic showing the sequence near the site ofCas9/gRNA gene editing within the HBB locus. Included is the sequencefor a wild type gene and for a sickle cell mutant gene. The sequence ofdifferent homology donor DNA templates that include either a sicklemutation or a sickle cell correction are shown. The donor DNA templatewith a sickle cell correction includes a β-thalassemia mutation.

FIGS. 22A-22B include bar graphs showing HDR editing efficiency forinsertion of donor DNA encoding a sickle cell mutation into the HBBlocus of CD34-expressing LT-HSPCs using AAV-mediated delivery of donorDNA. FIG. 22A shows a comparison of HDR efficiency for AAV given pre-EPor post-EP in combination with gRNA/Cas9 RNP. FIG. 22B shows acomparison of HDR efficiency in the presence of i53 or Nu7441 relativeto RNP+AAV-only.

FIGS. 23A-23B include dot plots showing % chimerism of human cellsderived from engrafted LT-HSPCs in mouse blood samples isolated at 8weeks and 16 weeks post-engraftment. FIG. 23A shows % chimerism forLT-HSPCs edited with gRNA/Cas9 RNP and AAV given either pre-EP orpost-EP. FIG. 23B shows % chimerism for LT-HSPCs edited with AAV andgRNA/Cas9 RNP in the presence of i53 or Nu7441 compared to RNP+AAV-only.

FIGS. 24A-24B include dot plots showing % chimerism of human cellsderived from engrafted LT-HSPCs in mouse bone marrow samples isolated at16 weeks post-engraftment. FIG. 24A shows % chimerism for LT-HSPCsedited with gRNA/Cas9 RNP and AAV given either pre-EP or post-EP. FIG.24B shows % chimerism for LT-HSPCs edited with AAV and gRNA/Cas9 RNP inthe presence of i53 or Nu7441 compared to RNP+AAV-only.

FIG. 25 includes a bar graph showing lineage distribution in mouse bonemarrow isolated at 16 weeks post-engraftment of LT-HSPCs. Shown is totalchimerism and percentage of human CD45-expressing cells that are Bcells, T cells, myeloid cells, or CD34-expressing hematopoieticstem/progenitor cells (HSPCs). Lineage distribution is shown forLT-HSPCs edited with gRNA/Cas9 RNP and AAV given either pre-EP orpost-EP. Also shown is lineage distribution for LT-HSPCs edited with AAVand gRNA/Cas9 RNP in the presence of i53 or Nu7441 compared toRNP+AAV-only.

FIGS. 26A-26B include dot plots showing HDR editing efficiency forinsertion of donor DNA encoding a sickle cell mutation into the HBBlocus, measured in mouse bone marrow isolated at 16 weekspost-engraftment of LT-HSPCs. FIG. 26A shows HDR editing efficiency forLT-HSPCs edited with gRNA/Cas9 RNP and AAV given either pre-EP orpost-EP. FIG. 26B shows HDR editing efficiency for LT-HSPCs edited withAAV and gRNA/Cas9 RNP in the presence of i53 or Nu7441 compared toRNP+AAV-only.

FIG. 27 includes a dot plot showing indel frequency in mouse bone marrowisolated at 16 weeks post-engraftment of LT-HSPCs relative to the indelfrequency of LT-HSPCs prior to engraftment (e.g., input indels). Shownis indel frequency for LT-HSPCs edited with gRNA/Cas9 RNP and AAV giveneither pre-EP or post-EP. Also shown is indel frequency for LT-HSPCsedited with AAV and gRNA/Cas9 RNP in the presence of i53 or Nu7441compared to RNP+AAV-only.

FIG. 28 includes a dot plot showing erythroid cell enucleation in mousebone marrow isolated at 16 weeks post-engraftment of LT-HSPCs. Shown isenucleation for LT-HSPCs edited with gRNA/Cas9 RNP and AAV given eitherpre-EP or post-EP. Also shown is enucleation for LT-HSPCs edited withAAV and gRNA/Cas9 RNP in the presence of i53 or Nu7441 compared toRNP+AAV-only.

FIG. 29 includes a schematic showing the sequence near the site ofCas9/gRNA gene editing within the HBB locus. Included is the sequencefor wild type HBB (healthy), for HBB encoding an E6V mutation (sickle),spacer sequence of R02 gRNA, and sequence of homology donor DNA encodedby AAV.323 to provide correction of the E6V sickle cell disease (SCD)mutation.

FIGS. 30A-30B provide bar graphs quantifying the frequency of a SCD genecorrection (E6V to E6) in HBB by HDR repair (FIG. 30A) and frequency ofINDELs in the HBB gene (FIG. 30B) in CD34-expressing LT-HSPCs derivedfrom a patient donor with SCD mutation that were subsequently edited byelectroporation with R02 gRNA/Cas9 RNP+AAV.323 in the presence of i53.Control cells were edited by electroporation with R02 gRNA/Cas9RNP+AAV.323 only, R02 gRNA/Cas9 RNP only, or without AAV or RNP (mockEP).

FIGS. 31A-31B provide bar graphs quantifying the frequency of SCD genecorrection by HDR repair (FIG. 31A) and frequency of INDELs in HBB (FIG.31B) measured either the same day as gene-editing (Day 0) or at 14 daysfollowing gene-editing and maintenance by in vitro culture (Day 14) forcells edited as in FIGS. 30A-30B.

FIG. 32A provides a bar graph quantifying the proportion of totalhemoglobin expressed by patient-derived CD34-expressing LT-HSPCs editedas in FIGS. 31A-31B that was HbF, HbA, or HbS as measured by HPLCanalysis. FIG. 32B provides an assessment of SCD correction forpatient-derived CD34-expressing LT-HSPCs edited with R02 gRNA/Cas9RNP+AAV.323+i53 that is comparison of the frequency of SCD genecorrection by HDR repair (“% HDR by NGS”) and percent decrease in HbSexpression relative to mock EP (no RNP or AAV) control cells (“% HbSdecrease by HPLC”).

FIGS. 33A-33B provide bar graphs quantifying the frequency of SCD genecorrection by HDR repair (FIG. 33A) and frequency of INDELs in HBB (FIG.33B) measured in PBMCSs or CD34-expressing LT-HSPCs isolated from apatient donor with SCD mutation that were subsequently edited byelectroporation with R02 gRNA/Cas9 RNP+AAV.323 in the presence of i53.Control cells were edited by electroporation with R02 gRNA/Cas9RNP+AAV.323 only, R02 gRNA/Cas9 RNP only, or without AAV or RNP (mockEP).

FIG. 34 provides a bar graph quantifying the proportion of totalhemoglobin expressed by patient-derived PBMCs or CD34-expressingLT-HSPCs edited as in FIGS. 33A-33B that was HbF, HbA, or HbS asmeasured by HPLC analysis.

FIGS. 35A-35C provide bar graphs quantifying HDR editing efficiency atthe R02 target site in the HBB gene (FIG. 35A) and the frequency ofINDELs at R02 off-target sites OT1 (FIG. 35B) and OT2 (FIG. 35C) forCD34-expressing LT-HSPCs edited with RNP containing wild-type (WT) Cas9or high fidelity (HiFi) Cas9 and R02 gRNA and AAV.307.

FIG. 36A provides a bar graph quantifying the ratio of beta globinmonomers (beta-globin (B), beta-globin with SCD mutation (S), andunknown beta-globin mutants (U)) to total hemoglobin expressed by SCDpatient and healthy donor-derived CD34+LT-HSPCs following editing and invitro differentiation. Cells were edited by electroporation with R02 RNPand AAV.323 or with R02 RNP only. Control cells were electroporatedwithout RNP or AAV (mock). FIG. 36B provides a bar graph quantifyingfold-change in total gamma-globin expression by cells edited as in FIG.36A compared to expression by mock control cells. FIG. 36C provides aschematic showing a 9 nt deletion in the HBB gene induced by repair ofan R02-induced DSB (NGS Read) as compared to the wild-type HBB gene(reference) and corresponding polypeptide sequence encoded by the NGSand reference reads.

FIGS. 37A-37B provide graphs showing HDR editing efficiency forinsertion of donor DNA encoding GFP in the HBB gene locus ofCD34-expressing LT-HSPCs following editing with R02 RNP+AAV-delivereddonor DNA encoding GFP either alone or in combination with a DNA-PKinhibitor (compound 296) provided at indicated concentrations. FIG. 37Afurther provides a measure of the percentage of total cells thatremained viable following editing. FIG. 37B further provides acomparison to cells edited with R02 RNP+AAV and mRNA encoding i53.

FIGS. 38A-38C provide graphs quantifying HDR editing efficiency andfrequency of INDELs in the HBB gene for CD34-expressing LT-HSPCs editedwith R02 RNP+AAV.307 either alone or in combination with compound 296 atindicated concentrations. Comparison is made to cells edited with R02RNP+AAV.307+i53 and control cells edited with R02 RNP only. FIGS.38A-38C represent data from independent experiments (“Experiment 1” or“Experiment 2”). FIG. 38C further provides a measure of the percentageof total cells that remained viable following editing. FIG. 38D providesa bar graph quantifying the percentage of total sequence reads having adeletion in HBB of 9 nt (corresponding to repair by the MMEJ pathway) oran INDEL in HBB of ±1 nt (corresponding to repair by NHEJ) for cellsedited as in FIGS. 38A-38C. The fold-reduction in INDEL frequency isindicated for cells edited with 10 μM compound 296 as compared to noediting with compound 296.

FIGS. 39A-39B provide graphs quantifying HDR editing efficiency andfrequency of INDELs in the HBB gene for CD34-expressing LT-HSPCs editedwith R02 RNP+AAV.307 either alone or in combination with the DNA-PKinhibitor compound 984 at indicated concentrations. Comparison is madeto control cells edited R02 RNP+AAV.307+i53 and control cells editedwith R02 RNP only.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the use of anunique donor polynucleotide designed to correct a Glu6Val (E6V) mutationin exon 1 of the HBB gene in combination with an efficient gRNA and asite-directed nuclease (e.g., SpCas9) to generate a double-strandedbreak (DSB) at a target site in HBB to correct the mutation via homologydirected repair (HDR), thereby treating sickle cell disease. The donorpolynucleotide comprises a nucleotide sequence that corrects the E6Vmutation and is homologous with a region of the HBB gene encoding theE6V mutation and all or part of target sequence for a site directednuclease (e.g., an SpCas9 PAM site or complement thereof). For example,the donor polynucleotide can include a nucleotide sequence that ishomologous with a region of the HBB gene that comprises a PAMrecognition site, or complement thereof, that is recognized by the sitedirected nuclease. In some embodiments, a recombinant vector comprisesthe donor polynucleotide that corrects the E6V mutation located betweentwo homology arms: a left homology arm (LHA) comprising a nucleotidesequence homologous to region in the HBB gene upstream of the E6Vmutation; and a right homology arm (RHA) comprising a nucleotidesequence homologous to a region in the HBB gene downstream of the E6Vmutation and spanning the target site. In some embodiments, the homologyarms are each about 500 nucleotides or more. In some embodiments, thehomology arms are each about 2.2 kb. In some aspects, the donorpolynucleotide is codon optimized to increase HDR.

The present disclosure is also based, at least in part, on the discoverythat CD34+ cells derived from sickle cell patients having the E6Vmutation in an HBB gene, were successfully genetically edited with theunique donor polynucleotide to correct the E6V mutation. It has beendemonstrated that such edited CD34+ cells differentiate into red bloodcells having the same characteristics (e.g., enucleation) as uneditedcells. It has also been shown that HDR of a DSB effected by the gRNA andDNA endonuclease was increased with a 53BP1 inhibitor and/or DNA-PKinhibitor. It has been further demonstrated that off-target activity ofa Cas9 endonuclease in combination with a gRNA targeting a target siteproximal to the E6V mutation is reduced by using a Cas9 endonucleaseengineered to have high-fidelity.

Genome Editing

In some aspects, the disclosure provides methods for editing a cell tocorrect an E6V mutation in human beta-globin (HBB). In some embodiments,the E6V mutation is corrected by editing an HBB gene encoding the E6Vmutation (i.e., genome editing). As is well known in the art and usedherein, the term “E7V” refers to a single nucleotide polymorphism (SNP)in the HBB gene that occurs in the seventh codon downstream thetranscription start site (i.e. the seventh codon of HBB if including theAUG start codon), wherein the SNP converts the wild-type codon encodingGlu to a codon encoding Val. Correspondingly, a beta-globin polypeptidewith an “E7V” mutation refers to substitution of Glu to Val occurring inthe seventh amino acid residue of the beta-globin polypeptide ifincluding the initial methionine amino acid. As used herein, the term“E6V” refers to a SNP in the HBB gene that occurs in the sixth codondownstream the AUG start codon (i.e., the sixth codon of the HBB openreading frame downstream the start codon), wherein the SNP converts thewild-type encoding Glu to a codon encoding Val. Correspondingly, abeta-globin polypeptide with an “E6V” mutation refers to substitution ofGlu to Val occurring at the sixth amino acid residue of the beta-globingpolypeptide, not including the initial methionine amino acid.Accordingly, as readily understood by one of ordinary skill in the art,the terms “E7V” and “E6V” refer to the same mutation in the HBB gene,and are used interchangeably herein when used in reference to the sicklemutation.

Genome editing generally refers to the process of editing or changingthe nucleotide sequence of a genome, preferably in a precise, desirableand/or pre-determined manner. Examples of compositions, systems, andmethods of genome editing described herein use site-directed nucleasesto cut or cleave DNA at precise target locations in the genome, therebycreating a double-strand break (DSB) in the DNA. Such breaks can berepaired by endogenous DNA repair pathways, such as homology directedrepair (HDR) and/or non-homologous end-joining (NHEJ) repair (see e.g.,Cox et al., (2015) Nature Medicine 21 (2):121-31). One of the majorobstacles to efficient genome editing in non-dividing cells is lack ofhomology directed repair (HDR). Without HDR, non-dividing cells rely onnon-homologous end joining (NHEJ) to repair double-strand breaks (DSB)that occur in the genome. The results of NHEJ-mediated DNA repair ofDSBs can include correct repair of the DSB, or deletion or insertion ofone or more nucleotides or polynucleotides.

In some embodiments, the disclosure provides improved methods forediting a cell to correct an E6V mutation encoded by the HBB gene. Insome embodiments, the disclosure provides methods for improving HDR of aDSB in a target region in an HBB gene. In some embodiments, the methodsdisclosed herein utilize a donor polynucleotide or recombinant vector, agRNA and a DNA endonuclease (e.g., SpCas9) to edit an HBB gene within acell (e.g., correct an E6V mutation encoded by the HBB gene). In someembodiments, the method disclosed herein utilize a donor polynucleotideor recombinant vector, a gRNA, a DNA endonuclease (e.g., SpCas9), and a53BP1 inhibitor and/or DNA-PKcs inhibitor, to improve genome editing ofan HBB gene within a cell (e.g., correction of an E6V mutation encodedby the HBB gene).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, and a Cas9endonuclease (e.g., SpCas9). In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98, a gRNAcomprising a spacer sequence corresponding to a target sequencecomprising SEQ ID NO: 15, and a Cas9 endonuclease (e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, and a Cas9 endonuclease (e.g., SpCas9). Insome embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, and a Cas9 endonuclease (e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 20, and a Cas9 endonuclease (e.g., SpCas9). Insome embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 20, and a Cas9 endonuclease (e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, and a Cas9 endonuclease (e.g., SpCas9). Insome embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising a spacer sequence comprisingSEQ ID NO: 16, and a Cas9 endonuclease (e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, and a Cas9endonuclease (e.g., SpCas9). In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98, a gRNAcomprising SEQ ID NO: 17, and a Cas9 endonuclease (e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence corresponding toa target sequence comprising SEQ ID NO: 15, and a high-fidelity Cas9endonuclease (e.g., high-fidelity SpCas9). In some embodiments, thedisclosure provides a donor polynucleotide or recombinant vector, a gRNAthat recognizes a target sequence comprising SEQ ID NO: 15, and ahigh-fidelity Cas9 endonuclease (e.g., high-fidelity SpCas9). In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA that recognizes a target sequence comprisingSEQ ID NO: 20, and a high-fidelity Cas9 endonuclease (e.g.,high-fidelity SpCas9). In some embodiments, the disclosure provides adonor polynucleotide or recombinant vector, a gRNA comprising a spacersequence comprising SEQ ID NO: 16, and a high-fidelity Cas9 endonuclease(e.g., high-fidelity SpCas9). In some embodiments, the disclosureprovides a donor polynucleotide or recombinant vector, a gRNA comprisingSEQ ID NO: 17, and a high-fidelity Cas9 endonuclease (e.g.,high-fidelity SpCas9).

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA targeting a target site in an HBBgene, and a Cas9 endonuclease (e.g., SpCas9). In some embodiments, thedisclosure provides a recombinant vector comprising SEQ ID NO: 98, agRNA targeting a target site in an HBB gene, and a Cas9 endonuclease(e.g., SpCas9).

In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence corresponding toa target sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9), and a 53BP1 inhibitor and/or DNA-PKcs inhibitor. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA that recognizes a target sequence comprisingSEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorand/or DNA-PKcs inhibitor. In some embodiments, the disclosure providesa donor polynucleotide or recombinant vector, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 20, a Cas9 endonuclease (e.g.,SpCas9), and a 53BP1 inhibitor and/or DNA-PKcs inhibitor. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence comprising SEQID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorand/or DNA-PKcs inhibitor. In some embodiments, the disclosure providesa donor polynucleotide or recombinant vector, a gRNA comprising SEQ IDNO: 17, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitor and/orDNA-PKcs inhibitor.

In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence corresponding toa target sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9), and a 53BP1 inhibitor comprising a polypeptide sequence of SEQID NO: 70 and/or DNA-PKcs inhibitor. In some embodiments, the disclosureprovides a donor polynucleotide or recombinant vector, a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9), and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcs inhibitor. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA that recognizes a target sequence comprisingSEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor. In some embodiments, the disclosure provides a donorpolynucleotide or recombinant vector, a gRNA comprising a spacersequence comprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9),and a 53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO: 70and/or DNA-PKcs inhibitor. In some embodiments, the disclosure providesa donor polynucleotide or recombinant vector, a gRNA comprising SEQ IDNO: 17, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor.

In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence corresponding toa target sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9), and a 53BP1 inhibitor and/or DNA-PKcs inhibitor selected fromCompound 984 and Compound 296. In some embodiments, the disclosureprovides a donor polynucleotide or recombinant vector, a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9), and a 53BP1 inhibitor and/or DNA-PKcsinhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA that recognizes a target sequence comprisingSEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorand/or DNA-PKcs inhibitor selected from Compound 984 and Compound 296.In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence comprising SEQID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorand/or DNA-PKcs inhibitor selected from Compound 984 and Compound 296.In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising SEQ ID NO: 17, a Cas9 endonuclease(e.g., SpCas9), and a 53BP1 inhibitor and/or DNA-PKcs inhibitor selectedfrom Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence corresponding toa target sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9), and a 53BP1 inhibitor comprising a polypeptide sequence of SEQID NO: 70 and/or DNA-PKcs inhibitor selected from Compound 984 andCompound 296. In some embodiments, the disclosure provides a donorpolynucleotide or recombinant vector, a gRNA that recognizes a targetsequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9),and a 53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO: 70and/or DNA-PKcs inhibitor selected from Compound 984 and Compound 296.In some embodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA that recognizes a target sequence comprisingSEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising a spacer sequence comprising SEQID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a donor polynucleotide orrecombinant vector, a gRNA comprising SEQ ID NO: 17, a Cas9 endonuclease(e.g., SpCas9), and a 53BP1 inhibitor comprising a polypeptide sequenceof SEQ ID NO: 70 and/or DNA-PKcs inhibitor selected from Compound 984and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA targeting a target site in an HBBgene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitor and/orDNA-PKcs inhibitor. In some embodiments, the disclosure provides arecombinant vector comprising SEQ ID NO: 98, a gRNA targeting a targetsite in an HBB gene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1inhibitor and/or DNA-PKcs inhibitor.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA targeting a target site in an HBBgene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA targeting a target site in anHBB gene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA targeting a target site in an HBBgene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitor and/orDNA-PKcs inhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a recombinant vector comprising SEQID NO: 98, a gRNA targeting a target site in an HBB gene, a Cas9endonuclease (e.g., SpCas9), and a 53BP1 inhibitor and/or DNA-PKcsinhibitor selected from Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA targeting a target site in an HBBgene, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcsinhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a recombinant vector comprising SEQID NO: 98, a gRNA targeting a target site in an HBB gene, a Cas9endonuclease (e.g., SpCas9), and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70 and/or DNA-PKcs inhibitor selectedfrom Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor. In some embodiments,the disclosure provides a donor polynucleotide comprising SEQ ID NO:102, a gRNA comprising a spacer sequence corresponding to a targetsequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9)and a 53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO:70. In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9) and a 53BP1inhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9) and a 53BP1 inhibitor comprising a polypeptide sequence of SEQID NO: 70. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 20, a Cas9 endonuclease (e.g.,SpCas9) and a 53BP1 inhibitor. In some embodiments, the disclosureprovides a donor polynucleotide comprising SEQ ID NO: 102, a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 20, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70. In some embodiments, thedisclosure provides a recombinant vector comprising SEQ ID NO: 98 a gRNAcomprising a spacer sequence corresponding to a target sequencecomprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9) and a 53BP1inhibitor. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70. In some embodiments, thedisclosure provides a recombinant vector comprising SEQ ID NO: 98 a gRNAthat recognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor. In some embodiments,the disclosure provides a recombinant vector comprising SEQ ID NO: 98, agRNA that recognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70. In some embodiments, thedisclosure provides a recombinant vector comprising SEQ ID NO: 98 a gRNAthat recognizes a target sequence comprising SEQ ID NO: 20, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor. In some embodiments,the disclosure provides a recombinant vector comprising SEQ ID NO: 98, agRNA that recognizes a target sequence comprising SEQ ID NO: 20, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a DNA-PKcs inhibitor. In someembodiments, the disclosure provides a donor polynucleotide comprisingSEQ ID NO: 102, a gRNA comprising a spacer sequence corresponding to atarget sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 and Compound296. In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9) and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 and Compound296. In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9) and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 20, a Cas9 endonuclease (e.g.,SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 and Compound296. In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9) and a DNA-PKcs inhibitor. In someembodiments, the disclosure provides a recombinant vector comprising SEQID NO: 98, a gRNA comprising a spacer sequence corresponding to a targetsequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9)and a DNA-PKcs inhibitor selected from Compound 984 and Compound 296. Insome embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9) and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides arecombinant vector comprising SEQ ID NO: 98, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 and Compound296. In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9) and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides arecombinant vector comprising SEQ ID NO: 98, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 20, a Cas9 endonuclease (e.g.,SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 and Compound296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecorresponding to a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor, and a DNA-PKcsinhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA comprising a spacersequence corresponding to a target sequence comprising SEQ ID NO: 15, aCas9 endonuclease (e.g., SpCas9), a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70 and a DNA-PKcs inhibitor selectedfrom Compound 984 and Compound 296. In some embodiments, the disclosureprovides a donor polynucleotide comprising SEQ ID NO: 102, a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor, and a DNA-PKcsinhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA that recognizes atarget sequence comprising SEQ ID NO: 15, a Cas9 endonuclease (e.g.,SpCas9), a 53BP1 inhibitor comprising a polypeptide sequence of SEQ IDNO: 70 and a DNA-PKcs inhibitor selected from Compound 984 and Compound296. In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9), a 53BP1inhibitor, and a DNA-PKcs inhibitor. In some embodiments, the disclosureprovides a donor polynucleotide comprising SEQ ID NO: 102, a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 20, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor comprising a polypeptidesequence of SEQ ID NO: 70 and a DNA-PKcs inhibitor selected fromCompound 984 and Compound 296. In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98 a gRNA comprisinga spacer sequence corresponding to a target sequence comprising SEQ IDNO: 15, a Cas9 endonuclease (e.g., SpCas9), a 53BP1 inhibitor and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides arecombinant vector comprising SEQ ID NO: 98, a gRNA comprising a spacersequence corresponding to a target sequence comprising SEQ ID NO: 15, aCas9 endonuclease (e.g., SpCas9), a 53BP1 inhibitor comprising apolypeptide sequence of SEQ ID NO: 70 and a DNA-PKcs inhibitor selectedfrom Compound 984 and Compound 296. In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98 a gRNA thatrecognizes a target sequence comprising SEQ ID NO: 15, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor and a DNA-PKcs inhibitor.In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA that recognizes a target sequencecomprising SEQ ID NO: 15, a Cas9 endonuclease (e.g., SpCas9), a 53BP1inhibitor comprising a polypeptide sequence of SEQ ID NO: 70 and aDNA-PKcs inhibitor selected from Compound 984 and Compound 296. In someembodiments, the disclosure provides a recombinant vector comprising SEQID NO: 98 a gRNA that recognizes a target sequence comprising SEQ ID NO:20, a Cas9 endonuclease (e.g., SpCas9), a 53BP1 inhibitor and a DNA-PKcsinhibitor. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA that recognizes a targetsequence comprising SEQ ID NO: 20, a Cas9 endonuclease (e.g., SpCas9), a53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO: 70 and aDNA-PKcs inhibitor selected from Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9) and a 53BP1inhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA comprising a spacersequence comprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9)and a 53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO:70. In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising a spacer sequence comprisingSEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a 53BP1inhibitor. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO: 70.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9) and aDNA-PKcs inhibitor. In some embodiments, the disclosure provides a donorpolynucleotide comprising SEQ ID NO: 102, a gRNA comprising a spacersequence comprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9)and a DNA-PKcs inhibitor selected from Compound 984 and Compound 296. Insome embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising a spacer sequence comprisingSEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and a DNA-PKcsinhibitor. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), and aDNA-PKcs inhibitor selected from Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising a spacer sequencecomprising SEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), a 53BP1inhibitor and a DNA-PKcs inhibitor. In some embodiments, the disclosureprovides a donor polynucleotide comprising SEQ ID NO: 102, a gRNAcomprising a spacer sequence comprising SEQ ID NO: 16, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor comprising a polypeptidesequence of SEQ ID NO: 70 and a DNA-PKcs inhibitor selected fromCompound 984 and Compound 296. In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98, a gRNAcomprising a spacer sequence comprising SEQ ID NO: 16, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor and a DNA-PKcs inhibitor.In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising a spacer sequence comprisingSEQ ID NO: 16, a Cas9 endonuclease (e.g., SpCas9), a 53BP1 inhibitorcomprising a polypeptide sequence of SEQ ID NO: 70, and a DNA-PKcsinhibitor selected from Compound 984 and Compound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9) and a 53BP1 inhibitor. In some embodiments,the disclosure provides a donor polynucleotide comprising SEQ ID NO:102, a gRNA comprising SEQ ID NO: 17, a Cas9 endonuclease (e.g., SpCas9)and a 53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO:70. In some embodiments, the disclosure provides a recombinant vectorcomprising SEQ ID NO: 98, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9), and a 53BP1 inhibitor. In some embodiments,the disclosure provides a recombinant vector comprising SEQ ID NO: 98, agRNA comprising SEQ ID NO: 17, a Cas9 endonuclease (e.g., SpCas9), and a53BP1 inhibitor comprising a polypeptide sequence of SEQ ID NO: 70.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9) and a DNA-PKcs inhibitor. In someembodiments, the disclosure provides a donor polynucleotide comprisingSEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, a Cas9 endonuclease(e.g., SpCas9) and a DNA-PKcs inhibitor selected from Compound 984 andCompound 296. In some embodiments, the disclosure provides a recombinantvector comprising SEQ ID NO: 98, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9), and a DNA-PKcs inhibitor. In someembodiments, the disclosure provides a recombinant vector comprising SEQID NO: 98, a gRNA comprising SEQ ID NO: 17, a Cas9 endonuclease (e.g.,SpCas9), and a DNA-PKcs inhibitor selected from Compound 984 andCompound 296.

In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor and a DNA-PKcs inhibitor.In some embodiments, the disclosure provides a donor polynucleotidecomprising SEQ ID NO: 102, a gRNA comprising SEQ ID NO: 17, a Cas9endonuclease (e.g., SpCas9), a 53BP1 inhibitor comprising a polypeptidesequence of SEQ ID NO: 70, and a DNA-PKcs inhibitor selected fromCompound 984 and Compound 296. In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98, a gRNAcomprising SEQ ID NO: 17, a Cas9 endonuclease (e.g., SpCas9), a 53BP1inhibitor and a DNA-PKcs inhibitor. In some embodiments, the disclosureprovides a recombinant vector comprising SEQ ID NO: 98, a gRNAcomprising SEQ ID NO: 17, a Cas9 endonuclease (e.g., SpCas9), a 53BP1inhibitor comprising a polypeptide sequence of SEQ ID NO: 70, and aDNA-PKcs inhibitor selected from Compound 984 and Compound 296.

In some embodiments, the donor polynucleotide comprises a nucleotidesequence complement to SEQ ID NO: 102. In some embodiments, therecombinant vector comprises a nucleotide sequence complement to SEQ IDNO: 98.

Donor Polynucleotides

The disclosure provides donor polynucleotides that, upon insertion intoa DSB, correct or induce a mutation in a target nucleic acid (e.g., agenomic DNA). In some embodiments, the donor polynucleotides provided bythe disclosure are recognized and used by the HDR machinery of a cell torepair a double strand break (DSB) introduced into a target nucleic acidby a site-directed nuclease, wherein repair of the DSB results in theinsertion of the donor polynucleotide into the target nucleic acid. Insome embodiments, the donor polynucleotides and/or recombinant vectorsprovided by the disclosure are recognized and used by the HDR machineryof a cell to repair a double strand break (DSB) introduced into a targetnucleic acid (e.g., HBB gene) by a site-directed nuclease, wherein theregion proximal to the DSB is exchanged for the corresponding regionprovided by the donor polynucleotide. Alternatively, a donorpolynucleotide may have no regions of homology to the targeted locationin the DNA and may be integrated by NHEJ-dependent end joining followingcleavage at the target site.

In some embodiments, a donor template can be DNA or RNA, single-strandedand/or double-stranded, and can be introduced into a cell in linear orcircular form. In some embodiments, the donor template can be a donorpolynucleotide or a recombinant vector. If introduced in linear form,the ends of the donor sequence can be protected (e.g., fromexonucleolytic degradation) by methods known to those of skill in theart. For example, one or more dideoxynucleotide residues are added tothe 3′ terminus of a linear molecule and/or self-complementaryoligonucleotides are ligated to one or both ends. See, for example,Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls etal., (1996) Science 272:886-889. Additional methods for protectingexogenous polynucleotides from degradation include, but are not limitedto, addition of terminal amino group(s) and the use of modifiedinternucleotide linkages such as, for example, phosphorothioates,phosphoramidates, and O-methyl ribose or deoxyribose residues.

In some embodiments, a donor template can be introduced into a cell aspart of a vector molecule having additional sequences such as, forexample, replication origins, promoters and genes encoding antibioticresistance. In some embodiments, a donor template can be introduced asnaked nucleic acid or as nucleic acid complexed with an agent such as aliposome or poloxamer. In some embodiments, a donor template or can bedelivered by a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus,lentivirus and integrase defective lentivirus (IDLV)).

A donor template, in some embodiments, is inserted so that itsexpression is driven by the endogenous promoter at the integration site,namely the promoter that drives expression of the endogenous gene intowhich the donor is inserted. In some embodiments, a donor template isintegrated so that its expression is driven by the endogenous promoterat the integration site, namely the promoter that drives expression ofthe endogenous gene into which the donor is exchanged. However, in someembodiments, the donor template comprises an exogenous promoter and/orenhancer, for example a constitutive promoter, an inducible promoter, ortissue-specific promoter. In some embodiments, the exogenous promoter isan EF1α promoter comprising a sequence of SEQ ID NO: 59. Other promotersknown to those of skill in the art may also be used.

In some embodiments, exogenous sequences may also includetranscriptional and/or translational regulatory sequences, for example,promoters, enhancers, insulators, internal ribosome entry sites,sequences encoding 2A peptides and/or polyadenylation signals.

In some embodiments, the donor polynucleotides comprise a nucleotidesequence which corrects or induces a mutation in a genomic DNA (gDNA)molecule in a cell, wherein when the donor polynucleotide is introducedinto the cell in combination with a site-directed nuclease, a HDR DNArepair pathway inserts the donor polynucleotide into a double-strandedDNA break (DSB) introduced into the gDNA by the site-directed nucleaseat a location proximal to the mutation, thereby correcting the mutation.In some embodiments, the donor polynucleotides and/or recombinantvectors comprise a nucleotide sequence which corrects or induces amutation in a genomic DNA (gDNA) molecule in a cell, wherein when thedonor polynucleotide is introduced into the cell in combination with asite-directed nuclease, a HDR DNA repair pathway exchanges a regionproximal to a double-stranded DNA break (DSB) for the correspondingregion provided by the donor polynucleotide and/or recombinant vectors,by the site-directed nuclease at a location proximal to the mutation,thereby correcting the mutation.

In some embodiments, the donor polynucleotide comprises a nucleotidesequence which corrects or induces a mutation, wherein the nucleotidesequence that corrects or induces a mutation comprises a singlenucleotide. In some embodiments, the nucleotide sequence which correctsor induces a mutation comprises two or more nucleotides. In someembodiments, the nucleotide sequence which corrects or induces amutation comprises a codon. In some embodiments, the nucleotide sequencewhich corrects or induces a mutation comprises one or more codons. Insome embodiments, the nucleotide sequence which corrects or induces amutation comprises an exonic sequence. In some embodiments, thenucleotide sequence which corrects or induces a mutation comprises anintronic sequence. In some embodiments, the nucleotide sequence whichcorrects or induces a mutation comprises all or a portion of an exonicsequence. In some embodiments, the nucleotide sequence which corrects orinduces a mutation comprises all or a portion of an intronic sequence.In some embodiments, the nucleotide sequence which corrects or induces amutation comprises all or a portion of an exonic sequence and all or aportion of an intronic sequence.

In some embodiments, the donor polynucleotide sequence is identical toor substantially identical to (having at least one nucleotidedifference) an endogenous sequence of a target nucleic acid. In someembodiments, the endogenous sequence comprises a genomic sequence of thecell. In some embodiments, the endogenous sequence comprises achromosomal or extrachromosomal sequence. In some embodiments, the donorpolynucleotide sequence comprises a sequence that is substantiallyidentical (comprises at least one nucleotide difference/change) to aportion of the endogenous sequence in a cell at or near the DSB. In someembodiments, repair of the target nucleic acid molecule with the donorpolynucleotide results in an insertion, deletion, or substitution of oneor more nucleotides of the target nucleic acid molecule. In someembodiments, the insertion, deletion, or substitution of one or morenucleotides results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the insertion, deletion, or substitution of one or morenucleotides results in one or more nucleotide changes in an RNAexpressed from the target gene. In some embodiments, the insertion,deletion, or substitution of one or more nucleotides alters theexpression level of the target gene. In some embodiments, the insertion,deletion, or substitution of one or more nucleotides results inincreased or decreased expression of the target gene. In someembodiments, the insertion, deletion, or substitution of one or morenucleotides results in gene knockdown. In some embodiments, theinsertion, deletion, or substitution of one or more nucleotides resultsin gene knockout. In some embodiments, the repair of the target nucleicacid molecule with the donor polynucleotide results in replacement of anexon sequence, an intron sequence, a transcriptional control sequence, atranslational control sequence, a sequence comprising a splicing signal,or a non-coding sequence of the target gene.

In some embodiments, the donor polynucleotide is of a suitable length tocorrect or induce a mutation in a gDNA. In some embodiments, the donorpolynucleotide comprises 10, 15, 20, 25, 50, 75, 100 or more nucleotidesin length. In some embodiments, the donor polynucleotide comprises 18nucleotides in length. In some embodiments, the donor polynucleotidecomprises 10-30 nucleotides in length. In some embodiments, the donorpolynucleotide comprises 10-20 nucleotides in length. In someembodiments, the donor polynucleotide comprises 15-25 nucleotides inlength. In some embodiments, the donor polynucleotide comprises 20-30nucleotides in length. In some embodiments, the donor polynucleotidecomprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 nucleotides in length.

In some embodiments, the donor polynucleotide comprises a nucleotidesequence homologous to a region in a target gene. In some embodiments,the nucleotide sequence homologous to a region in a target gene is10-30, 10-20, 15-25 or 20-30 nucleotides in length.

In some embodiments (for example those described herein where a donorpolynucleotide is incorporated into the cleaved nucleic acid as aninsertion mediated by non-homologous end joining) the donorpolynucleotide has no homology arms. In some embodiments, to facilitateHDR repair of a DSB, the donor polynucleotide has flanking homology arms(for example those described herein where a donor polynucleotide isincorporated into the cleaved nucleic acid as an insertion mediated byHDR repair). In some embodiments, the donor polynucleotide is about10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length.In some embodiments, the donor polynucleotide is about 10-100nucleotides in length. In some embodiments, the donor polynucleotide isabout 20-80 nucleotides in length. In some embodiments, the donorpolynucleotide is about 30-70 nucleotides in length. In someembodiments, the donor polynucleotide is about 40-60 nucleotides inlength. In some embodiments, the donor polynucleotide is 40, 41, 42, 43,44, 45, 46, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60nucleotides in length. In some embodiments, the donor polynucleotide is40 nucleotides in length. In some embodiments, the donor polynucleotideis 41 nucleotides in length. In some embodiments, the donorpolynucleotide is 42 nucleotides in length. In some embodiments, thedonor polynucleotide is 43 nucleotides in length. In some embodiments,the donor polynucleotide is 44 nucleotides in length. In someembodiments, the donor polynucleotide is 45 nucleotides in length. Insome embodiments, the donor polynucleotide is 46 nucleotides in length.In some embodiments, the donor polynucleotide is 47 nucleotides inlength. In some embodiments, the donor polynucleotide is 48 nucleotidesin length. In some embodiments, the donor polynucleotide is 49nucleotides in length. In some embodiments, the donor polynucleotide is50 nucleotides in length. In some embodiments, the donor polynucleotideis 51 nucleotides in length. In some embodiments, the donorpolynucleotide is 52 nucleotides in length. In some embodiments, thedonor polynucleotide is 53 nucleotides in length. In some embodiments,the donor polynucleotide is 54 nucleotides in length. In someembodiments, the donor polynucleotide is 55 nucleotides in length. Insome embodiments, the donor polynucleotide is 56 nucleotides in length.In some embodiments, the donor polynucleotide is 57 nucleotides inlength. In some embodiments, the donor polynucleotide is 58 nucleotidesin length. In some embodiments, the donor polynucleotide is 59nucleotides in length. In some embodiments, the donor polynucleotide is60 nucleotides in length.

In some embodiments, a donor polynucleotide comprising exogenous geneticmaterial is flanked by homology arms to allow integration of theexogenous genetic material by HDR repair of a DSB in a target gene. Thehomology arms are designed to anneal to regions of gDNA that flank a DSBin a target gene. Methods of designing homology arms that allow HDRrepair of a DSB site in a target gene are taught in the art. See forexample US 20110281361 which is incorporated by reference herein.

In some embodiments, for HDR repair of a DSB, a donor polynucleotidecomprises a left and right flanking homology arms (LHA and RHA) thatallow annealing to gDNA. In some embodiments, the homology arms flankthe mutation or correction being introduced at the site of a DSB. Insome embodiments, a recombinant vector comprises the donorpolynucleotide flanked by a LHA and a RHA. In some embodiments, thehomology arms are at least 30-100, at least 50-200, at least 100-300, atleast 100-500, at least 250-1000, at least 500-1500 nucleotides inlength. In some embodiments, the homology arms are at least 100nucleotides in length. In some embodiments, the homology arms are atleast 200-500, at least 450-1000, at least 500-1500, at least 1000-2000,at least 1500-2500, at least 2000-3000, or at least 2500-3500nucleotides in length. In some embodiments, the homology arms are atleast 200 nucleotides in length. In some embodiments, the homology armsare at least 300 nucleotides in length. In some embodiments, thehomology arms are at least 400 nucleotides in length. In someembodiments, the homology arms are at least 500 nucleotides in length.In some embodiments, the homology arms are at least 600 nucleotides inlength. In some embodiments, the homology arms are at least 700nucleotides in length. In some embodiments, the homology arms are atleast 800 nucleotides in length. In some embodiments, the homology armsare at least 900 nucleotides in length. In some embodiments, thehomology arms are at least 1000 nucleotides in length. In someembodiments, the homology arms are at least 1500 nucleotides in length.In some embodiments, the homology arms are at least 2000 nucleotides inlength. In some embodiments, the homology arms are at least 2500nucleotides in length. In some embodiments, the homology arms are atleast 3000 nucleotides in length. In some embodiments, the homology armsare at least 3500 nucleotides in length.

In some embodiments, the LHA is at least 200-500, at least 450-1000, atleast 500-1500, at least 1000-2000, at least 1500-2500, at least2000-3000, or at least 2500-3500 nucleotides in length. In someembodiments, the LHA is about 500 to about 2500 nucleotides in length.In some embodiments, the LHA is about 2.2 kb. In some embodiments, theRHA is at least 200-500, at least 450-1000, at least 500-1500, at least1000-2000, at least 1500-2500, at least 2000-3000, or at least 2500-3500nucleotides in length. In some embodiments, the RHA is about 500 toabout 2500 nucleotides in length. In some embodiments, the RHA is about2.2 kb.

In some embodiments, the LHA and the RHA are the same length. In someembodiments, the LHA and the RHA are different lengths. In someembodiments, the LHA and the RHA have a combined length of about 400 toabout 5000, about 500 to about 4500, about 1000 to about 4400nucleotides. In some embodiments, the LHA and the RHA have a combinedlength of about 4400 nucleotides. In some embodiments, the LHA is about500 to about 2500 nucleotides in length and the RHA is about 500 toabout 2500 nucleotides in length. In some embodiments, the LHA is about2.2 kb in length and the RHA is about 2.2 kb in length. The rate of HDRis a function of the distance between the mutation at the DSB cut site.Thus, in some embodiments, the homology arms are designed to anneal togDNA directly adjacent to the site of a DSB. In some embodiments, a leftor right homology arm is designed to anneal to gDNA no more than 1-10nucleotides, 5-15, 10-30, 15-40, or 15-50 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 1 nucleotide from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 2 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 3 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 4 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 5 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 6 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 7 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 8 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 9 nucleotides from the DSB sitein a target gene. In some embodiments, a left or right homology arm isdesigned to anneal to gDNA no more than 10 nucleotides from the DSB sitein a target gene.

In some embodiments, the homology arms of a donor polynucleotide arefully complementary to gDNA flanking a DSB site in a target gene. Insome embodiments, the homology arms of a donor polynucleotide havesufficient complementary to gDNA flanking a DSB site in a target gene toallow HDR repair. In some embodiments, the homology arms within arecombinant vector are fully complementary to gDNA flanking a DSB sitein a target gene. In some embodiments, the homology arms within arecombinant vector have sufficient complementary to gDNA flanking a DSBsite in a target gene to allow HDR repair.

In some embodiments, a donor polynucleotide provided by the disclosurecomprises an intronic sequence. In some embodiments, the donorpolynucleotide comprises an intronic sequence which corrects or inducesa mutation in a gDNA. In some embodiments, the donor polynucleotidecomprises an exonic sequence. In some embodiments, the donorpolynucleotide comprises an exonic sequence which corrects or induces amutation in a gDNA.

Donor Polynucleotide Correcting SCD Mutation

In some embodiments, the disclosure provides a donor polynucleotideand/or recombinant vector that corrects an SCD mutation (e.g., E6V/E7Vin exon 1 of the HBB gene) in a cell. In some embodiments, the donorpolynucleotide comprises GAG or GAA to correct the GTG codon that leadsto the E6V mutation. In some embodiments, the donor polynucleotidecomprises a polynucleotide sequence having at least about 90% identifyto the nucleotide sequence set forth in SEQ ID NO: 102, or a complementthereof. In some embodiments, the disclosure provides a donorpolynucleotide can include a nucleotide sequence that is homologous witha region of the HBB gene that comprises a PAM recognition site, orcomplement thereof, that is recognized by the site directed nuclease. Insome embodiments, the disclosure provides a donor polynucleotide and/orrecombinant vector that mutates the PAM recognition site in the targetsequence to ensure that the site directed nuclease does not cleave thedonor polynucleotide after it is exchanged. In some embodiments, the PAMrecognition site is mutated to a polynucleotide sequence withoutintroducing a single nucleotide polymorphism (SNP) associated withβ-thalassemia. In some embodiments, the donor polynucleotide comprisesthe nucleotide sequence set forth in SEQ ID NO: 102, or a complementthereof. In some embodiments, the donor polynucleotide is codonoptimized to improve HDR.

In some embodiments, the donor polynucleotide is 10-20 bases, 15-25bases, 20-30 bases, 25-35 bases, or 30-40 bases in length. In someembodiments, the donor polynucleotide is 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 bases in length.

In some embodiments, the donor polynucleotide is located between twohomology arms (LHA and RHA). In some embodiments, the LHA and RHA arethe same length. In some embodiments, the LHA and RHA are differentlengths. In some embodiments, the homology arms are each about 500bases, about 600 bases, about 700 bases, about 800 bases, about 900bases, about 1 kb, about 1.5 kb, about 2 kb, about 2.5 kb, or about 3 kbin length. In some embodiments, the homology arms are each about 1.1 kb,1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb,2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kbor 3 kb in length. In some embodiments, the LHA is about 0.5 kb to about3 kb in length, and the RHA is about 0.5 kb to about 3 kb in length,wherein the LHA and RHA are different lengths. In some embodiments, theLHA is about 0.5 kb to about 3 kb in length, and the RHA is about 0.5 kbto about 3 kb in length, wherein the LHA and RHA are the same length. Insome embodiments, the LHA is about 0.5 kb to about 1.5 kb, about 1.0 kbto about 2.0 kb, about 1.5 kb to about 2.5 kb, or about 2.0 kb to about3.0 kb, and the RHA is about 0.5 kb to about 1.5 kb, about 1.0 kb toabout 2.0 kb, about 1.5 kb to about 2.5 kb, or about 2.0 kb to about 3.0kb, wherein the LHA and RHA are different lengths. In some embodiments,the LHA is about 0.5 kb to about 1.5 kb, about 1.0 kb to about 2.0 kb,about 1.5 kb to about 2.5 kb, or about 2.0 kb to about 3.0 kb, and theRHA is about 0.5 kb to about 1.5 kb, about 1.0 kb to about 2.0 kb, about1.5 kb to about 2.5 kb, or about 2.0 kb to about 3.0 kb, wherein the LHAand RHA are the same length. In some embodiments, the LHA is about 2.2kb and the RHA is about 2.2 kb. In some embodiments, the length of eachhomology arm is determined based on the capacity of the delivery systemused to provide the donor polynucleotide.

In some embodiments, the LHA comprises a nucleotide sequence homologousor substantially homologous to exon 1 of the HBB gene. In someembodiments, the LHA comprises a nucleotide sequence homologous orsubstantially homologous to a region upstream of an E6V mutation in exon1 of the HBB gene. In some embodiments, the LHA comprises a nucleotidesequence homologous or substantially homologous to the promoter for theHBB gene. In some embodiments, the LHA comprises a nucleotide sequencehomologous or substantially homologous to regions upstream of the HBBgene. In some embodiments, the LHA comprises a nucleotide sequencehomologous or substantially homologous to a region of exon 1 of the HBBgene upstream of an E6V mutation, along with the promoter and regionsupstream of the HBB gene. In some embodiments, the LHA comprises thenucleotide sequence set forth in SEQ ID NO: 99. In some embodiments, theLHA comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identity to SEQ ID NO: 99.

In some embodiments, the LHA comprises a nucleotide sequence homologousor substantially homologous to exon 1 of the HBB gene and is about 0.5kb to about 3.0 kb. In some embodiments, the LHA comprises a nucleotidesequence homologous or substantially homologous to a region upstream ofan E6V mutation in exon 1 of the HBB gene and is about 0.5 kb to about3.0 kb. In some embodiments, the LHA comprises a nucleotide sequencehomologous or substantially homologous to the promoter for the HBB geneand is about 0.5 kb to about 3.0 kb. In some embodiments, the LHAcomprises a nucleotide sequence homologous or substantially homologousto regions upstream of the HBB gene and is about 0.5 kb to about 3.0 kb.In some embodiments, the LHA comprises a nucleotide sequence homologousor substantially homologous to a region of exon 1 of the HBB geneupstream of an E6V mutation, along with the promoter and regionsupstream of the HBB gene and is about 0.5 kb to about 3.0 kb. In someembodiments, the LHA comprises the nucleotide sequence set forth in SEQID NO: 99. In some embodiments, the LHA comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 99.

In some embodiments, the RHA comprises a nucleotide sequence homologousor substantially homologous to exon 1 of the HBB gene. In someembodiments, the RHA comprises a nucleotide sequence homologous orsubstantially homologous to a region downstream of an E6V mutation inexon 1 of the HBB gene. In some embodiments, the RHA comprises anucleotide sequence homologous or substantially homologous to a regiondownstream of a double-strand break (DSB) effected by a gRNA andendonuclease. In some embodiments, the RHA comprises a nucleotidesequence that spans the target site. In some embodiments, the RHAcomprises a nucleotide sequence homologous or substantially homologousto all or a portion of intron 1-2 of the HBB gene. In some embodiments,the RHA comprises a nucleotide sequence homologous or substantiallyhomologous to all or a portion of exon 2 of the HBB gene. In someembodiments, the RHA comprises a nucleotide sequence homologous orsubstantially homologous to all or a portion of intron 2-3 of the HBBgene. In some embodiments, the RHA comprises a nucleotide sequencehomologous or substantially homologous to all or a portion of exon 3 ofthe HBB gene. In some embodiments, the RHA comprises a nucleotidesequence homologous or substantially homologous to a region downstreamof the DSB in exon 1, intron 1-2, exon 2, and a portion of intron 2-3,inclusive. In some embodiments, the RHA comprises a nucleotide sequencehomologous or substantially homologous to a region downstream of the DSBin exon 1, intron 1-2, exon 2, intron 2-3, and a portion of exon 3,inclusive. In some embodiments, the RHA comprises a nucleotide sequencehomologous or substantially homologous to a region downstream of the DSBin exon 1, intron 1-2, exon 2, intron 2-3, and exon 3 inclusive. In someembodiments, the RHA comprises the nucleotide sequence set forth in SEQID NO: 100. In some embodiments, the RHA comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 100.

In some embodiments, the RHA comprises a nucleotide sequence homologousor substantially homologous to exon 1 of the HBB gene and is about 0.5kb to about 3.0 kb. In some embodiments, the RHA comprises a nucleotidesequence homologous or substantially homologous to a region downstreamof an E6V mutation in exon 1 of the HBB gene and is about 0.5 kb toabout 3.0 kb. In some embodiments, the RHA comprises a nucleotidesequence homologous or substantially homologous to a region downstreamof a double-strand break (DSB) effected by a gRNA and endonuclease, andis about 0.5 kb to about 3.0 kb In some embodiments, the RHA comprises anucleotide sequence that spans the target site, and is about 0.5 kb toabout 3.0 kb. In some embodiments, the RHA comprises a nucleotidesequence homologous or substantially homologous to all or a portion ofintron 1-2 of the HBB gene and is about 0.5 kb to about 3.0 kb. In someembodiments, the RHA comprises a nucleotide sequence homologous orsubstantially homologous to all or a portion of exon 2 of the HBB geneand is about 0.5 kb to about 3.0 kb. In some embodiments, the RHAcomprises a nucleotide sequence homologous or substantially homologousto all or a portion of intron 2-3 of the HBB gene and is about 0.5 kb toabout 3.0 kb. In some embodiments, the RHA comprises a nucleotidesequence homologous or substantially homologous to all or a portion ofexon 3 of the HBB gene and is about 0.5 kb to about 3.0 kb. In someembodiments, the RHA comprises a nucleotide sequence homologous orsubstantially homologous to a region downstream of the DSB in exon 1,intron 1-2, exon 2, and a portion of intron 2-3, inclusive and is about0.5 kb to about 3.0 kb. In some embodiments, the RHA comprises anucleotide sequence homologous or substantially homologous to a regiondownstream of the DSB in exon 1, intron 1-2, exon 2, intron 2-3, and aportion of exon 3, inclusive and is about 0.5 kb to about 3.0 kb. Insome embodiments, the RHA comprises a nucleotide sequence homologous orsubstantially homologous to a region downstream of the DSB in exon 1,intron 1-2, exon 2, intron 2-3, and exon 3 inclusive and is about 0.5 kbto about 3.0 kb. In some embodiments, the RHA comprises the nucleotidesequence set forth in SEQ ID NO: 100. In some embodiments, the RHAcomprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identity to SEQ ID NO: 100.

In some embodiments, the disclosure provides a recombinant vectorcomprising a donor polynucleotide located between an LHA and an RHA, therecombinant vector having about 400 bases, about 500 bases, about 600bases, about 700 bases, about 800 bases, about 900 bases, about 1 kb,about 1.5 kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about4 kb, or about 4.5 kb in length. In some embodiments, the nucleotidesequence is about 2.5 kb, about 2.6 kb, about 2.7 kb, about 2.8 kb,about 2.9 kb, about 3 kb, about 3.1 kb, about 3.2 kb, about 3.3 kb,about 3.4 kb, about 3.5 kb, about 3.6 kb, about 3.7 kb, about 3.8 kb,about 3.9 kb, about 4 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb,about 4.4 kb or about 4.5 kb in length. In some embodiments, therecombinant vector is about 4.2 kb in length.

In some embodiments, the nucleotide sequence and/or recombinant vectorcomprising a donor polynucleotide located between an LHA and an RHAcomprises the sequence set forth in SEQ ID NO: 98. In some embodiments,the nucleotide sequence and/or recombinant vector comprises a nucleotidesequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 98.

Methods of Making and Testing Donor Polynucleotides

The donor polynucleotides provided by the disclosure are produced bysuitable DNA synthesis method or means known in the art. Recombinantvectors can also be produced by said methods. DNA synthesis is thenatural or artificial creation of deoxyribonucleic acid (DNA) molecules.The term DNA synthesis refers to DNA replication, DNA biosynthesis(e.g., in vivo DNA amplification), enzymatic DNA synthesis (e.g.,polymerase chain reaction (PCR); in vitro DNA amplification) or chemicalDNA synthesis.

In some embodiments, each strand of the donor polynucleotide is producedby oligonucleotide synthesis. Oligonucleotide synthesis is the chemicalsynthesis of relatively short fragments or strands of single-strandednucleic acids with a defined chemical structure (sequence). Methods ofoligonucleotide synthesis are known in the art (see e.g., Reese (2005)Organic & Biomolecular Chemistry 3(21):3851). The two strands can thenbe annealed together or duplexed to form a donor polynucleotide.

In some aspects, the insertion of a donor polynucleotide into a DSB isdetermined by a suitable method known in the art. For example, after theinsertional event, the nucleotide sequence of PCR amplicons generatedusing PCR primer that flank the DSB site is analyzed for the presence ofthe nucleotide sequence comprising the donor polynucleotide. In someembodiments, next-generation sequencing (NGS) techniques are used todetermine the extent of donor polynucleotide insertion into a DSBanalyzing PCR amplicons for the presence or absence of the donorpolynucleotide sequence. Further, since each donor polynucleotide is alinear, dsDNA molecule, which can insert in either of two orientations,NGS analysis can be used to determine the extent of insertion of thedonor polynucleotide in either direction.

In some aspects, the insertion of the donor polynucleotide and itsability to correct a mutation is determined by nucleotide sequenceanalysis of mRNA transcribed from the gDNA into which the donorpolynucleotide is inserted. An mRNA transcribed from gDNA containing aninserted donor polynucleotide is analyzed by a suitable method known inthe art. For example, conversion of mRNA extracted from cells treated orcontacted with a donor polynucleotide or system provided by thedisclosure is enzymatically converted into cDNA, which is further byanalyzed by NGS analysis to determine the extent of mRNA moleculecomprising the corrected mutation.

In other aspects, the insertion of a donor polynucleotide and itsability to correct a mutation is determined by protein sequence analysisof a polypeptide translated from an mRNA transcribed from the gDNA intowhich the donor polynucleotide is inserted. In some embodiments, a donorpolynucleotide corrects or induces a mutation by the incorporation of acodon into an exon that makes an amino acid change in a gene comprisinga gDNA molecule, wherein translation of an mRNA from the gene containingthe inserted donor polynucleotide generates a polypeptide comprising theamino acid change. The amino acid change in the polypeptide isdetermined by protein sequence analysis using techniques including, butnot limited to, Sanger sequencing, mass spectrometry, functional assaysthat measure an enzymatic activity of the polypeptide, or immunoblottingusing an antibody reactive to the amino acid change.

Use of Donor Polynucleotides to Correct or Induce a Mutation

In some embodiments, a donor polynucleotide provided by the disclosureis used to correct or induce a mutation in a gDNA in a cell by insertionof the donor polynucleotide into a target nucleic acid (e.g., gDNA) at acleavage site (e.g., a DSB) induced by a site-directed nuclease, such asthose described herein. In some embodiments, a donor polynucleotideprovided by the disclosure is used to correct or induce a mutation in agDNA in a cell by exchanging a region proximal to a cleavage site (e.g.,a DSB) for the corresponding region provided by the donor polynucleotidein a target nucleic acid (e.g., gDNA), induced by a site-directednuclease, such as those described herein. In some embodiments, HDR DNArepair mechanisms of the cell repair the DSB using the donorpolynucleotide, thereby inserting the donor polynucleotide into the DSBand adding the nucleotide sequence of the donor polynucleotide to thegDNA. In some embodiments, HDR DNA repair mechanisms of the cell repairthe DSB use the donor polynucleotide, thereby exchanging a region in thegDNA for the corresponding region provided by the donor polynucleotide,thus adding the nucleotide sequence of the donor polynucleotide to thegDNA. In some embodiments, the donor polynucleotide comprises anucleotide sequence which corrects a disease-causing mutation in a gDNAin a cell. In some embodiments, the donor polynucleotide is inserted ata location proximal to the mutation, thereby correcting the mutation. Insome embodiments, the donor polynucleotide is exchanged at a locationproximal to the mutation, thereby correcting the mutation. In someembodiments, the mutation is a substitution, missense, nonsense,insertion, deletion or frameshift mutation. In some embodiments themutation is in an exon. In some embodiments, the mutation is asubstitution, insertion or deletion and is located in an intron. In someembodiments, the mutation is proximal to a cleavage site in a gDNA. Insome embodiments, the mutation is a protein-coding mutation. In someembodiments, the mutation is associated with or causes a disease.

In some embodiments, the donor polynucleotide is inserted into the DSBby HDR DNA repair. In some embodiments, the donor polynucleotide isexchanged a location proximal to the DSB by HDR DNA repair. In someembodiments, the donor polynucleotide, a portion of the donorpolynucleotide is inserted into the target nucleic acid cleavage site byHDR DNA repair. In some embodiments, the donor polynucleotide, a portionof the donor polynucleotide is exchanged proximal to a target nucleicacid cleavage site by HDR DNA repair. In certain aspects, insertion of adonor polynucleotide into the target nucleic acid via HDR repair canresult in, for example, mutations, deletions, alterations, integrations,gene correction, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation of the endogenous gene sequence. In certain aspects, exchangeof a donor polynucleotide into the target nucleic acid via HDR repaircan result in, for example, mutations, deletions, alterations,integrations, gene correction, gene replacement, gene tagging, transgeneinsertion, nucleotide deletion, gene disruption, translocations and/orgene mutation of the endogenous gene sequence.

In some embodiments, the disease-causing mutation in the HBB generesults in an E6V amino acid substitution in the human beta-globinprotein. In some embodiments, the donor polynucleotide comprises anucleotide sequence which corrects a E6V mutation encoded by an HBB genein a gDNA in a cell. In some embodiments, the disclosure provides donorpolynucleotides used to repair a DSB introduced into a target nucleicacid molecule (e.g., gDNA) by a site-directed nuclease (e.g., Cas9) in acell. In some embodiments, the disclosure provides donor polynucleotidesused to repair a DSB introduced into an HBB gene by Cas9 in a cell. Insome embodiments, the donor polynucleotide is used by the HDR repairpathway of the cell to repair the DSB in the target nucleic acidmolecule. In some embodiments, the donor polynucleotide is used by theHDR repair pathway of the cell to repair the DSB in the HBB gene. Insome embodiments, the site-directed nuclease is a Cas nuclease. In someembodiments, the Cas nuclease is Cas9. The site-directed nucleasesdescribed herein can introduce DSB in target nucleic acids (e.g.,genomic DNA) in a cell. The introduction of a DSB in the genomic DNA ofa cell, induced by a site-directed nuclease, will stimulate theendogenous DNA repair pathways, such as those described herein. The HDRpathway can be used to insert a polynucleotide (e.g., a donorpolynucleotide) into the DSB during repair.

Accordingly, in some embodiments, a single donor polynucleotide ormultiple copies of the same donor polynucleotide are provided. In otherembodiments, two or more donor polynucleotides are provided such thatrepair may occur at two or more target sites. For example, differentdonor polynucleotides are provided to repair a single gene in a cell, ortwo different genes in a cell. In some embodiments, the different donorpolynucleotides are provided in independent copy numbers.

In some embodiments, the donor polynucleotide is incorporated into thetarget nucleic acid as an insertion mediated by HDR. In someembodiments, the donor polynucleotide sequence has no similarity to thenucleic acid sequence near the cleavage site. In some embodiments, asingle donor polynucleotide or multiple copies of the same donorpolynucleotide are provided. In other embodiments, two or more donorpolynucleotides having different sequences are inserted at two or moresites by non-homologous end joining. In some embodiments, the differentdonor polynucleotides are provided in independent copy numbers.

Systems for Genome Editing

In some aspects, the disclosure provide systems for correcting amutation in a genomic DNA molecule. In some embodiments, the systemcomprises a site-directed nuclease, such as a CRISPR/Cas system andoptionally a gRNA, and a donor polynucleotide, such as those describedherein. In some embodiments of the present disclosure, the systemcomprises an engineered nuclease. In some embodiments, the systemcomprises a site-directed nuclease. In some embodiments, thesite-directed nuclease comprises a CRISPR/Cas nuclease system. In someembodiments, the Cas nuclease is Cas9. In some embodiments, the guideRNA comprising the CRISPR/Cas system is an sgRNA.

CRISPR/Cas Nuclease Systems

Naturally-occurring CRISPR/Cas systems are genetic defense systems thatprovides a form of acquired immunity in prokaryotes. CRISPR is anabbreviation for Clustered Regularly Interspaced Short PalindromicRepeats, a family of DNA sequences found in the genomes of bacteria andarchaea that contain fragments of DNA (spacer DNA) with similarity toforeign DNA previously exposed to the cell, for example, by viruses thathave infected or attacked the prokaryote. These fragments of DNA areused by the prokaryote to detect and destroy similar foreign DNA uponre-introduction, for example, from similar viruses during subsequentattacks. Transcription of the CRISPR locus results in the formation ofan RNA molecule comprising the spacer sequence, which associates withand targets Cas (CRISPR-associated) proteins able to recognize and cutthe foreign, exogenous DNA. Numerous types and classes of CRISPR/Cassystems have been described (see e.g., Koonin et al., (2017) Curr OpinMicrobiol 37:67-78).

Engineered versions of CRISPR/Cas systems has been developed in numerousformats to mutate or edit genomic DNA of cells from other species. Thegeneral approach of using the CRISPR/Cas system involves theheterologous expression or introduction of a site-directed nuclease(e.g.: Cas nuclease) in combination with a guide RNA (gRNA) into a cell,resulting in a DNA cleavage event (e.g., the formation a single-strandor double-strand break (SSB or DSB)) in the backbone of the cell'sgenomic DNA at a precise, targetable location. The manner in which theDNA cleavage event is repaired by the cell provides the opportunity toedit the genome by the addition, removal, or modification (substitution)of DNA nucleotide(s) or sequences (e.g., genes).

Guide RNAs (gRNAs)

Engineered CRISPR/Cas systems comprise at least two components: 1) aguide RNA (gRNA) molecule and 2) a Cas nuclease, which interact to forma gRNA/Cas nuclease complex. A gRNA comprises at least a user-definedtargeting domain termed a “spacer” comprising a nucleotide sequence anda CRISPR repeat sequence. In engineered CRISPR/Cas systems, a gRNA/Casnuclease complex is targeted to a specific target sequence of interestwithin a target nucleic acid (e.g., a genomic DNA molecule) bygenerating a gRNA comprising a spacer with a nucleotide sequence that isable to bind to the specific target sequence in a complementary fashion(See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al.,Nature, 471, 602-607 (2011)). Thus, the spacer provides the targetingfunction of the gRNA/Cas nuclease complex.

In naturally-occurring type II-CRISPR/Cas systems, the “gRNA” iscomprised of two RNA strands: 1) a CRISPR RNA (crRNA) comprising thespacer and CRISPR repeat sequence, and 2) a trans-activating CRISPR RNA(tracrRNA). In Type II-CRISPR/Cas systems, the portion of the crRNAcomprising the CRISPR repeat sequence and a portion of the tracrRNAhybridize to form a crRNA:tracrRNA duplex, which interacts with a Casnuclease (e.g., Cas9). As used herein, the terms “split gRNA” or“modular gRNA” refer to a gRNA molecule comprising two RNA strands,wherein the first RNA strand incorporates the crRNA function(s) and/orstructure and the second RNA strand incorporates the tracrRNAfunction(s) and/or structure, and wherein the first and second RNAstrands partially hybridize.

Accordingly, in some embodiments, a gRNA provided by the disclosurecomprises two RNA molecules. In some embodiments, the gRNA comprises aCRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In someembodiments, the gRNA is a split gRNA. In some embodiments, the gRNA isa modular gRNA. In some embodiments, the split gRNA comprises a firststrand comprising, from 5′ to 3′, a spacer, and a first region ofcomplementarity; and a second strand comprising, from 5′ to 3′, a secondregion of complementarity; and optionally a tail domain.

In some embodiments, the crRNA comprises a spacer comprising anucleotide sequence that is complementary to and hybridizes with asequence that is complementary to the target sequence on a targetnucleic acid (e.g., a genomic DNA molecule). In some embodiments, thecrRNA comprises a region that is complementary to and hybridizes with aportion of the tracrRNA.

In some embodiments, the tracrRNA may comprise all or a portion of awild-type tracrRNA sequence from a naturally-occurring CRISPR/Cassystem. In some embodiments, the tracrRNA may comprise a truncated ormodified variant of the wild-type tracr RNA. The length of the tracr RNAmay depend on the CRISPR/Cas system used. In some embodiments, thetracrRNA may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100nucleotides in length. In certain embodiments, the tracrRNA is at least26 nucleotides in length. In additional embodiments, the tracrRNA is atleast 40 nucleotides in length. In some embodiments, the tracrRNA maycomprise certain secondary structures, such as, e.g., one or morehairpins or stem-loop structures, or one or more bulge structures.

Single Guide RNA (sgRNA)

Engineered CRISPR/Cas nuclease systems often combine a crRNA and atracrRNA into a single RNA molecule, referred to herein as a “singleguide RNA” (sgRNA), by adding a linker between these components. Withoutbeing bound by theory, similar to a duplexed crRNA and tracrRNA, ansgRNA will form a complex with a Cas nuclease (e.g., Cas9), guide theCas nuclease to a target sequence and activate the Cas nuclease forcleavage the target nucleic acid (e.g., genomic DNA). Accordingly, insome embodiments, the gRNA may comprise a crRNA and a tracrRNA that areoperably linked. In some embodiments, the sgRNA may comprise a crRNAcovalently linked to a tracrRNA. In some embodiments, the crRNA and thetracrRNA is covalently linked via a linker. In some embodiments, thesgRNA may comprise a stem-loop structure via base pairing between thecrRNA and the tracrRNA. In some embodiments, a sgRNA comprises, from 5′to 3′, a spacer, a first region of complementarity, a linking domain, asecond region of complementarity, and, optionally, a tail domain.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. The sgRNA can comprise a less than 20 nucleotidespacer sequence at the 5′ end of the sgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a variable length spacer sequencewith 17-30 nucleotides at the 5′ end of the sgRNA sequence as set forthby SEQ ID NO: 1.

The sgRNA can comprise no uracil at the 3′ end of the sgRNA sequence.The sgRNA can comprise one or more uracil at the 3′ end of the sgRNAsequence. For example, the sgRNA can comprise 1 uracil (U) at the 3′ endof the sgRNA sequence. The sgRNA can comprise 2 uracil (UU) at the 3′end of the sgRNA sequence. The sgRNA can comprise 3 uracil (UUU) at the3′ end of the sgRNA sequence. The sgRNA can comprise 4 uracil (UUUU) atthe 3′ end of the sgRNA sequence. The sgRNA can comprise 5 uracil(UUUUU) at the 3′ end of the sgRNA sequence. The sgRNA can comprise 6uracil (UUUUUU) at the 3′ end of the sgRNA sequence. The sgRNA cancomprise 7 uracil (UUUUUUU) at the 3′ end of the sgRNA sequence. ThesgRNA can comprise 8 uracil (UUUUUUUUU) at the 3′ end of the sgRNAsequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides.

In some embodiments, the sgRNA comprises a spacer sequence comprisingSEQ ID NO: 16. In some embodiments, the sgRNA comprises SEQ ID NO: 17.In some embodiments, the sgRNA comprises a nucleotide sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO 17.

By way of illustration, guide RNAs used in the CRISPR/Cas system, orother smaller RNAs can be readily synthesized by chemical means, asillustrated herein and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 endonuclease, are more readilygenerated enzymatically. Various types of RNA modifications can beintroduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

Spacers

In some embodiments, the gRNAs provided by the disclosure comprise aspacer sequence. A spacer sequence is a sequence that defines the targetsite of a target nucleic acid (e.g.: DNA). The target nucleic acid is adouble-stranded molecule: one strand comprises the target sequenceadjacent to a PAM sequence and is referred to as the “PAM strand,” andthe second strand is referred to as the “non-PAM strand” and iscomplementary to the PAM strand and target sequence. Both gRNA spacerand the target sequence are complementary to the non-PAM strand of thetarget nucleic acid. In some embodiments, a spacer sequencecorresponding to a target sequence adjacent to a PAM sequence iscomplementary to the non-PAM strand of the target nucleic acid. Thus, insome embodiments, a spacer sequence which corresponds to a targetsequence adjacent to a PAM sequence is identical to the PAM strand. ThegRNA spacer sequence hybridizes to the complementary strand (e.g.: thenon-PAM strand of the target nucleic acid/target site). In someembodiments, the spacer is sufficiently complementary to thecomplementary strand of the target sequence (e.g.: non-PAM strand), asto target a Cas nuclease to the target nucleic acid. In someembodiments, the spacer is at least 80%, 85%, 90% or 95% complementaryto the non-PAM strand of the target nucleic acid. In some embodiments,the spacer is 100% complementary to the non-PAM strand of the targetnucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6or more nucleotides that are not complementary with the non-PAM strandof the target nucleic acid. In some embodiments, the spacer comprises 1nucleotide that is not complementary with the non-PAM strand of thetarget nucleic acid. In some embodiments, the spacer comprises 2nucleotides that are not complementary with the non-PAM strand of thetarget nucleic acid.

In some embodiments, the 5′ most nucleotide of gRNA comprises the 5′most nucleotide of the spacer. In some embodiments, the spacer islocated at the 5′ end of the crRNA. In some embodiments, the spacer islocated at the 5′ end of the sgRNA. In some embodiments, the spacer isabout 15-50, about 20-45, about 25-40 or about 30-35 nucleotides inlength. In some embodiments, the spacer is about 19-22 nucleotides inlength. In some embodiments the spacer is about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In someembodiments the spacer is 19 nucleotides in length. In some embodiments,the spacer is 20 nucleotides in length, in some embodiments, the spaceris 21 nucleotides in length.

In some embodiments, the nucleotide sequence of the target sequence andthe PAM comprises the formula 5′ N19-21-N-R-G-3′ (SEQ ID NO: 63),wherein N is any nucleotide, and wherein R is a nucleotide comprisingthe nucleobase adenine (A) or guanine (G), and wherein the three 3′terminal nucleic acids, N-R-G represent the S. pyogenes PAM (SEQ ID NO:64). In some embodiments, the nucleotide sequence of the spacer isdesigned or chosen using a computer program. The computer program canuse variables, such as predicted melting temperature, secondarystructure formation, predicted annealing temperature, sequence identity,genomic context, chromatin accessibility, % GC, frequency of genomicoccurrence (e.g., of sequences that are identical or are similar butvary in one or more spots as a result of mismatch, insertion ordeletion), methylation status, and/or presence of SNPs.

In some embodiments, the spacer comprise at least one or more modifiednucleotide(s) such as those described herein. The disclosure providesgRNA molecules comprising a spacer which may comprise the nucleobaseuracil (U), while any DNA encoding a gRNA comprising a spacer comprisingthe nucleobase uracil (U) will comprise the nucleobase thymine (T) inthe corresponding position(s).

In some embodiments, the spacer sequence corresponds to a targetsequence comprising SEQ ID NO: 15. In some embodiments, the spacersequence corresponds to a target sequence comprising SEQ ID NO: 15 andcomprises 1, 2, 3, 4, 5, 6 or more nucleotides that are notcomplementary with the non-PAM strand of the target nucleic acid.

In some embodiments, the spacer sequence comprises SEQ ID NO: 16. Insome embodiments, the spacer sequence comprises a sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16.

Methods of Making gRNAs

The gRNAs of the present disclosure is produced by a suitable meansavailable in the art, including but not limited to in vitrotranscription (IVT), synthetic and/or chemical synthesis methods, or acombination thereof. Enzymatic (IVT), solid-phase, liquid-phase,combined synthetic methods, small region synthesis, and ligation methodsare utilized. In one embodiment, the gRNAs are made using IVT enzymaticsynthesis methods. Methods of making polynucleotides by IVT are known inthe art and are described in International Application PCT/US2013/30062.Accordingly, the present disclosure also includes polynucleotides, e.g.,DNA, constructs and vectors are used to in vitro transcribe a gRNAdescribed herein.

In some aspects, non-natural modified nucleobases are introduced intopolynucleotides, e.g., gRNA, during synthesis or post-synthesis. Incertain embodiments, modifications are on internucleoside linkages,purine or pyrimidine bases, or sugar. In particular embodiments, themodification is introduced at the terminal of a polynucleotide; withchemical synthesis or with a polymerase enzyme. Examples of modifiednucleic acids and their synthesis are disclosed in PCT application No.PCT/US2012/058519. Synthesis of modified polynucleotides is alsodescribed in Verma and Eckstein, Annual Review of Biochemistry, vol. 76,99-134 (1998).

In some aspects, enzymatic or chemical ligation methods are used toconjugate polynucleotides or their regions with different functionalmoieties, such as targeting or delivery agents, fluorescent labels,liquids, nanoparticles, etc. Conjugates of polynucleotides and modifiedpolynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

Certain embodiments of the invention also provide nucleic acids, e.g.,vectors, encoding gRNAs described herein. In some embodiments, thenucleic acid is a DNA molecule. In other embodiments, the nucleic acidis an RNA molecule. In some embodiments, the nucleic acid comprises anucleotide sequence encoding a crRNA. In some embodiments, thenucleotide sequence encoding the crRNA comprises a spacer flanked by allor a portion of a repeat sequence from a naturally-occurring CRISPR/Cassystem. In some embodiments, the nucleic acid comprises a nucleotidesequence encoding a tracrRNA. In some embodiments, the crRNA and thetracrRNA is encoded by two separate nucleic acids. In other embodiments,the crRNA and the tracrRNA is encoded by a single nucleic acid. In someembodiments, the crRNA and the tracrRNA is encoded by opposite strandsof a single nucleic acid. In other embodiments, the crRNA and thetracrRNA is encoded by the same strand of a single nucleic acid.

In some embodiments, the gRNAs provided by the disclosure are chemicallysynthesized by any means described in the art (see e.g., WO/2005/01248).While chemical synthetic procedures are continually expanding,purifications of such RNAs by procedures such as high performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach used forgenerating RNAs of greater length is to produce two or more moleculesthat are ligated together.

In some embodiments, the gRNAs provided by the disclosure aresynthesized by enzymatic methods (e.g., in vitro transcription, IVT).

Various types of RNA modifications can be introduced during or afterchemical synthesis and/or enzymatic generation of RNAs, e.g.,modifications that enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

In certain embodiments, more than one guide RNA can be used with aCRISPR/Cas nuclease system. Each guide RNA may contain a differenttargeting sequence, such that the CRISPR/Cas system cleaves more thanone target nucleic acid. In some embodiments, one or more guide RNAs mayhave the same or differing properties such as activity or stabilitywithin the Cas9 RNP complex. Where more than one guide RNA is used, eachguide RNA can be encoded on the same or on different vectors. Thepromoters used to drive expression of the more than one guide RNA is thesame or different.

The guide RNA may target any sequence of interest via the targetingsequence (e.g.: spacer sequence) of the crRNA. In some embodiments, thedegree of complementarity between the targeting sequence of the guideRNA and the target sequence on the target nucleic acid molecule is about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule is 100% complementary. Inother embodiments, the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule may contain at leastone mismatch. For example, the targeting sequence of the guide RNA andthe target sequence on the target nucleic acid molecule may contain 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, thetargeting sequence of the guide RNA and the target sequence on thetarget nucleic acid molecule may contain 1-6 mismatches. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule may contain 5 or 6mismatches.

The length of the targeting sequence may depend on the CRISPR/Cas9system and components used. For example, different Cas9 proteins fromdifferent bacterial species have varying optimal targeting sequencelengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. Insome embodiments, the targeting sequence may comprise 18-24 nucleotidesin length. In some embodiments, the targeting sequence may comprise19-21 nucleotides in length. In some embodiments, the targeting sequencemay comprise 20 nucleotides in length.

In some embodiments of the present disclosure, a CRISPR/Cas nucleasesystem includes at least one guide RNA. In some embodiments, the guideRNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., aCRISPR/Cas complex. The guide RNA may guide the Cas protein to a targetsequence on a target nucleic acid molecule (e.g., a genomic DNAmolecule), where the Cas protein cleaves the target nucleic acid. Insome embodiments, the CRISPR/Cas complex is a Cpf1/guide RNA complex. Insome embodiments, the CRISPR complex is a Type-II CRISPR/Cas9 complex.In some embodiments, the Cas protein is a Cas9 protein. In someembodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex.

Cas Nuclease

In some embodiments, the disclosure provides compositions and systems(e.g., an engineered CRISPR/Cas system) comprising a site-directednuclease, wherein the site-directed nuclease is a Cas nuclease. The Casnuclease may comprise at least one domain that interacts with a guideRNA (gRNA). Additionally, the Cas nuclease are directed to a targetsequence by a guide RNA. The guide RNA interacts with the Cas nucleaseas well as the target sequence such that, once directed to the targetsequence, the Cas nuclease is capable of cleaving the target sequence.In some embodiments, the guide RNA provides the specificity for thecleavage of the target sequence, and the Cas nuclease are universal andpaired with different guide RNAs to cleave different target sequences.

In some embodiments, the CRISPR/Cas system comprise components derivedfrom a Type-I, Type-II, or Type-III system. Updated classificationschemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cassystems, having Types I to V or VI (Makarova et al., (2015) Nat RevMicrobiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).Class 2 CRISPR/Cas systems have single protein effectors. Cas proteinsof Types II, V, and VI are single-protein, RNA-guided endonucleases,herein called “Class 2 Cas nucleases.” Class 2 Cas nucleases include,for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9,and contains a RuvC-like nuclease domain.

In some embodiments, the Cas nuclease are from a Type-II CRISPR/Cassystem (e.g., a Cas9 protein from a CRISPR/Cas9 system). In someembodiments, the Cas nuclease are from a Class 2 CRISPR/Cas system (asingle-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein).The Cas9 and Cpf1 family of proteins are enzymes with DNA endonucleaseactivity, and they can be directed to cleave a desired nucleic acidtarget by designing an appropriate guide RNA, as described furtherherein.

A Type-II CRISPR/Cas system component are from a Type-IIA, Type-IIB, orType-IIC system. Cas9 and its orthologs are encompassed. Non-limitingexemplary species that the Cas9 nuclease or other components are frominclude Streptococcus pyogenes, Streptococcus thermophilus,Streptococcus sp., Staphylococcus aureus, Listeria innocua,Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes,Sutterella wadsworthensis, Gamma proteobacterium, Neisseriameningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobactersuccinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei,Streptomyces pristinaespiralis, Streptomyces viridochromogenes,Streptomyces viridochromogenes, Streptosporangium roseum,Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacilluspseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum,Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillusbuchneri, Treponema denticola, Microscilla marina, Burkholderialesbacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaerawatsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.,Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptorbecscii, Candidatus Desulforudis, Clostridium botulinum, Clostridiumdifficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculumthermopropionicum, Acidithiobacillus caldus, Acidithiobacillusferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcushalophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis,Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaenavariabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima,Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleuschthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosiphoafricanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacterlari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, orAcaryochloris marina. In some embodiments, the Cas9 protein are fromStreptococcus pyogenes (SpCas9). In some embodiments, the Cas9 proteinare from Streptococcus thermophilus (StCas9). In some embodiments, theCas9 protein are from Neisseria meningitides (NmCas9). In someembodiments, the Cas9 protein are from Staphylococcus aureus (SaCas9).In some embodiments, the Cas9 protein are from Campylobacter jejuni(CjCas9).

In some embodiments, a Cas nuclease may comprise more than one nucleasedomain. For example, a Cas9 nuclease may comprise at least one RuvC-likenuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain(e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB inthe target sequence. In some embodiments, the Cas9 nuclease is modifiedto contain only one functional nuclease domain. For example, the Cas9nuclease is modified such that one of the nuclease domains is mutated orfully or partially deleted to reduce its nucleic acid cleavage activity.In some embodiments, the Cas9 nuclease is modified to contain nofunctional RuvC-like nuclease domain. In other embodiments, the Cas9nuclease is modified to contain no functional HNH-like nuclease domain.In some embodiments in which only one of the nuclease domains isfunctional, the Cas9 nuclease is a nickase that is capable ofintroducing a single-stranded break (a “nick”) into the target sequence.In some embodiments, a conserved amino acid within a Cas9 nucleasenuclease domain is substituted to reduce or alter a nuclease activity.In some embodiments, the Cas nuclease nickase comprises an amino acidsubstitution in the RuvC-like nuclease domain. Exemplary amino acidsubstitutions in the RuvC-like nuclease domain include D10A (based onthe S. pyogenes Cas9 nuclease). In some embodiments, the nickasecomprises an amino acid substitution in the HNH-like nuclease domain.Exemplary amino acid substitutions in the HNH-like nuclease domaininclude E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenesCas9 nuclease). In some embodiments, the nuclease system describedherein comprises a nickase and a pair of guide RNAs that arecomplementary to the sense and antisense strands of the target sequence,respectively. The guide RNAs directs the nickase to target and introducea DSB by generating a nick on opposite strands of the target sequence(i.e., double nicking). Chimeric Cas9 nucleases are used, where onedomain or region of the protein is replaced by a portion of a differentprotein. For example, a Cas9 nuclease domain is replaced with a domainfrom a different nuclease such as Fok1. A Cas9 nuclease is a modifiednuclease.

In alternative embodiments, the Cas nuclease is from a Type-I CRISPR/Cassystem. In some embodiments, the Cas nuclease is a component of theCascade complex of a Type-I CRISPR/Cas system. For example, the Casnuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease isderived from a Type-III CRISPR/Cas system. In some embodiments, the Casnuclease is derived from Type-IV CRISPR/Cas system. In some embodiments,the Cas nuclease is derived from a Type-V CRISPR/Cas system. In someembodiments, the Cas nuclease is derived from a Type-VI CRISPR/Cassystem.

High Fidelity Endonucleases

In some embodiments, the disclosure provides a CRISPR/Cas systemcomprising a Cas nuclease engineered for increased fidelity. As usedherein, the term “fidelity” when used in reference to a CRISPR/Cassystem comprising a Cas nuclease and gRNA refers to the specificity ofthe system for a target site in a DNA molecule (e.g., genomic DNAmolecule) that is homologous (e.g., perfect match) to the gRNA spacersequence. In some embodiments, a CRISPR/Cas system with increasedfidelity has reduced activity at off-target sites in the DNA molecule,i.e., sites that are an imperfect match to the gRNA spacer sequence.

In some embodiments, a CRISPR/Cas system of the disclosure comprises aCas variant comprising one or more mutations for increased fidelity. Insome embodiments, the one or more mutations result in reduced activityof the CRISPR/Cas system at off-target sites in the DNA molecule, forexample, compared to a system comprising an unmodified version of theCas nuclease (e.g., wild-type Cas nuclease). In some embodiments, theCRISPR/Cas system has substantially equivalent activity for inducingcleavage at an on-target site in the DNA molecule, for example, ascompared to the system comprising an unmodified version of the Casnuclease.

Methods of making Cas variants with increased fidelity are known in theart. For example, in some embodiments, a method of structure-guidedengineering is used to make a Cas variant with increased fidelity.

In some embodiments, a CRISPR/Cas system described herein comprises aCas9 nuclease comprising one or more mutations for increased fidelity.In some embodiments, the Cas9 nuclease is derived from S. pyogenes,wherein the Cas nuclease comprises one or more mutations relative towild-type SpCas9 for increased fidelity.

A suitable Cas9 nuclease with increased fidelity for use in the presentdisclosure includes any one described US2019/0010471; US2018/0142222;U.S. Pat. No. 9,944,912; WO2020/057481; US2019/0177710; US2018/0100148;U.S. Pat. No. 10,526,591; and US20200149020; each of which isincorporated herein by reference in their entirety.

In some embodiments, a Cas nuclease engineered for increased fidelityreduces cleavage of one or more predicted off-target sites by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 100%, at least about 110%, at least about 115%, at leastabout 120%, at least about 125%, at least about 30%, at least about135%, at least about 140%, at least about 145%, at least about 150%, atleast about 155%, at least about 160%, at least about 165%, at leastabout 170%, at least about 175%, at least about 180%, at least about185%, at least about 190%, at least about 195%, or at least about 200%,relative to a Cas nuclease not engineered for increased fidelity (e.g.,wild-type Cas nuclease). In some embodiments, a Cas nuclease engineeredfor increased fidelity reduces cleavage of one or more predictedoff-target sites by about 10% to about 200%, about 20% to about 190%,about 30% to about 180%, about 40% to about 170%, about 50% to about160%, about 60% to about 150%, about 70% to about 140%, about 80% toabout 130%, about 90% to about 120%, about 100% to about 110%, relativeto a Cas nuclease not engineered for increased fidelity (e.g., wild-typeCas nuclease).

In some embodiments, cleavage of an off-target or on-target site isdetermined based on the percentage of INDELs. In some embodiments, thepercentage of INDELs generated at one or more off-target sites by a Casnuclease engineered for increased fidelity is decreased relative to thepercentage of INDELs generated by a Cas nuclease not engineered forincreased fidelity (e.g., wild-type Cas nuclease).

In some embodiments, a Cas nuclease engineered for increased fidelitymaintains the same level of cleavage of the on-target site, and reducesthe cleavage of one or more predicted off-target sites compared to a Casnuclease not engineered for increased fidelity (e.g., wild-type Casnuclease).

Engineered Nucleases

In additional embodiments, the donor polynucleotides provided by thedisclosure are used in combination with a site-directed nuclease,wherein the site-directed nuclease is an engineered nuclease. Exemplaryengineered nucleases are meganuclease (e.g., homing endonucleases), ZFN,TALEN, and megaTAL.

Naturally-occurring meganucleases may recognize and cleavedouble-stranded DNA sequences of about 12 to 40 base pairs and arecommonly grouped into five families. In some embodiments, themeganuclease are chosen from the LAGLIDADG family, the GIY-YIG family,the HNH family, the His-Cys box family, and the PD-(D/E)XK family. Insome embodiments, the DNA binding domain of the meganuclease areengineered to recognize and bind to a sequence other than its cognatetarget sequence. In some embodiments, the DNA binding domain of themeganuclease are fused to a heterologous nuclease domain. In someembodiments, the meganuclease, such as a homing endonuclease, are fusedto TAL modules to create a hybrid protein, such as a “megaTAL” protein.The megaTAL protein have improved DNA targeting specificity byrecognizing the target sequences of both the DNA binding domain of themeganuclease and the TAL modules.

ZFNs are fusion proteins comprising a zinc-finger DNA binding domain(“zinc fingers” or “ZFs”) and a nuclease domain. Eachnaturally-occurring ZF may bind to three consecutive base pairs (a DNAtriplet), and ZF repeats are combined to recognize a DNA target sequenceand provide sufficient affinity. Thus, engineered ZF repeats arecombined to recognize longer DNA sequences, such as, e.g., 9-, 12-, 15-,or 18-bp, etc. In some embodiments, the ZFN comprise ZFs fused to anuclease domain from a restriction endonuclease. For example, therestriction endonuclease is FokI. In some embodiments, the nucleasedomain comprises a dimerization domain, such as when the nucleasedimerizes to be active, and a pair of ZFNs comprising the ZF repeats andthe nuclease domain is designed for targeting a target sequence, whichcomprises two half target sequences recognized by each ZF repeats onopposite strands of the DNA molecule, with an interconnecting sequencein between (which is sometimes called a spacer in the literature). Forexample, the interconnecting sequence is 5 to 7 bp in length. When bothZFNs of the pair bind, the nuclease domain may dimerize and introduce aDSB within the interconnecting sequence. In some embodiments, thedimerization domain of the nuclease domain comprises a knob-into-holemotif to promote dimerization. For example, the ZFN comprises aknob-into-hole motif in the dimerization domain of FokI.

The DNA binding domain of TALENs usually comprises a variable number of34 or 35 amino acid repeats (“modules” or “TAL modules”), with eachmodule binding to a single DNA base pair, A, T, G, or C. Adjacentresidues at positions 12 and 13 (the “repeat-variable di-residue” orRVD) of each module specify the single DNA base pair that the modulebinds to. Though modules used to recognize G may also have affinity forA, TALENs benefit from a simple code of recognition—one module for eachof the 4 bases—which greatly simplifies the customization of aDNA-binding domain recognizing a specific target sequence. In someembodiments, the TALEN may comprise a nuclease domain from a restrictionendonuclease. For example, the restriction endonuclease is FokI. In someembodiments, the nuclease domain may dimerize to be active, and a pairof TALENS is designed for targeting a target sequence, which comprisestwo half target sequences recognized by each DNA binding domain onopposite strands of the DNA molecule, with an interconnecting sequencein between. For example, each half target sequence is in the range of 10to 20 bp, and the interconnecting sequence is 12 to 19 bp in length.When both TALENs of the pair bind, the nuclease domain may dimerize andintroduce a DSB within the interconnecting sequence. In someembodiments, the dimerization domain of the nuclease domain may comprisea knob-into-hole motif to promote dimerization. For example, the TALENmay comprise a knob-into-hole motif in the dimerization domain of FokI.

Modified Nucleases

In certain embodiments, the nuclease is optionally modified from itswild-type counterpart. In some embodiments, the nuclease is fused withat least one heterologous protein domain. At least one protein domain islocated at the N-terminus, the C-terminus, or in an internal location ofthe nuclease. In some embodiments, two or more heterologous proteindomains are at one or more locations on the nuclease.

In some embodiments, the protein domain may facilitate transport of thenuclease into the nucleus of a cell. For example, the protein domain isa nuclear localization signal (NLS). In some embodiments, the nucleaseis fused with 1-10 NLS(s). In some embodiments, the nuclease is fusedwith 1-5 NLS(s). In some embodiments, the nuclease is fused with oneNLS. In other embodiments, the nuclease is fused with more than one NLS.In some embodiments, the nuclease is fused with 2, 3, 4, or 5 NLSs. Insome embodiments, the nuclease is fused with 2 NLSs. In someembodiments, the nuclease is fused with 3 NLSs. In some embodiments, thenuclease is fused with no NLS. In some embodiments, the NLS may be amonopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO:65) or PKKKRRV (SEQ ID NO: 66). In some embodiments, the NLS is abipartite sequence, such as, e.g., the NLS of nucleoplasmin,KRPAATKKAGQAKKKK (SEQ ID NO: 67). In some embodiments, the NLS isgenetically modified from its wild-type counterpart.

In some embodiments, the protein domain is capable of modifying theintracellular half-life of the nuclease. In some embodiments, thehalf-life of the nuclease may be increased. In some embodiments, thehalf-life of the nuclease is reduced. In some embodiments, the entity iscapable of increasing the stability of the nuclease. In someembodiments, the entity is capable of reducing the stability of thenuclease. In some embodiments, the protein domain act as a signalpeptide for protein degradation. In some embodiments, the proteindegradation is mediated by proteolytic enzymes, such as, e.g.,proteasomes, lysosomal proteases, or calpain proteases. In someembodiments, the protein domain comprises a PEST sequence. In someembodiments, the nuclease is modified by addition of ubiquitin or apolyubiquitin chain. In some embodiments, the ubiquitin is aubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-likeproteins include small ubiquitin-like modifier (SUMO), ubiquitincross-reactive protein (UCRP, also known as interferon-stimulatedgene-15 (ISG15)), ubiquitin-related modifier-1 (URM1),neuronal-precursor-cell-expressed developmentally downregulatedprotein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyteantigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fauubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitinfold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).

In some embodiments, the protein domain is a marker domain. Non-limitingexamples of marker domains include fluorescent proteins, purificationtags, epitope tags, and reporter gene sequences. In some embodiments,the marker domain is a fluorescent protein. Non-limiting examples ofsuitable fluorescent proteins include green fluorescent proteins (e.g.,GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green,Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescentproteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl),blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv,Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean,CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate,mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2,DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry,mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO,Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or anyother suitable fluorescent protein. In other embodiments, the markerdomain is a purification tag and/or an epitope tag. Non-limitingexemplary tags include glutathione-S-transferase (GST), chitin bindingprotein (CBP), maltose binding protein (MBP), thioredoxin (TRX),poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS,E, ECS, E2, FLAG (SEQ ID NO: 95), HA, nus, Softag 1, Softag 3, Strep,SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His (SEQ ID NO: 94),biotin carboxyl carrier protein (BCCP), and calmodulin. Non-limitingexemplary reporter genes include glutathione-S-transferase (GST),horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT),beta-galactosidase, beta-glucuronidase, luciferase, or fluorescentproteins.

In additional embodiments, the protein domain may target the nuclease toa specific organelle, cell type, tissue, or organ.

In further embodiments, the protein domain is an effector domain. Whenthe nuclease is directed to its target nucleic acid, e.g., when a Cas9protein is directed to a target nucleic acid by a guide RNA, theeffector domain may modify or affect the target nucleic acid. In someembodiments, the effector domain is chosen from a nucleic acid bindingdomain, a nuclease domain, an epigenetic modification domain, atranscriptional activation domain, or a transcriptional repressordomain. In some embodiments, the effector domain can be a nucleobasedeaminase domain.

Certain embodiments of the invention also provide nucleic acids encodingthe nucleases (e.g., a Cas9 protein) described herein provided on avector. In some embodiments, the nucleic acid is a DNA molecule. Inother embodiments, the nucleic acid is an RNA molecule. In someembodiments, the nucleic acid encoding the nuclease is an mRNA molecule.In certain embodiments, the nucleic acid is an mRNA encoding a Cas9protein.

In some embodiments, the nucleic acid encoding the nuclease is codonoptimized for efficient expression in one or more eukaryotic cell types.In some embodiments, the nucleic acid encoding the nuclease is codonoptimized for efficient expression in one or more mammalian cells. Insome embodiments, the nucleic acid encoding the nuclease is codonoptimized for efficient expression in human cells. Methods of codonoptimization including codon usage tables and codon optimizationalgorithms are available in the art.

Target Sites

In some embodiments, the site-directed nucleases described herein aredirected to and cleave (e.g., introduce a DSB) a target nucleic acidmolecule. In some embodiments, the target nucleic acid molecule is anHBB gene. In some embodiments, a Cas nuclease is directed by a guide RNAto a target site of a target nucleic acid molecule (gDNA), where theguide RNA hybridizes with the complementary strand of the targetsequence and the Cas nuclease cleaves the target nucleic acid at thetarget site. In some embodiments, a Cas nuclease is directed by a gRNAto a target site of an HBB gene. In some embodiments, the Cas nucleaseis directed by a gRNA to a target site comprising SEQ ID NO: 15 or 20.In some embodiments, the complementary strand of the target sequence iscomplementary to the targeting sequence (e.g.: spacer sequence) of theguide RNA. In some embodiments, the degree of complementarity between atargeting sequence of a guide RNA and its corresponding complementarystrand of the target sequence is about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, thecomplementary strand of the target sequence and the targeting sequenceof the guide RNA is 100% complementary. In other embodiments, thecomplementary strand of the target sequence and the targeting sequenceof the guide RNA contains at least one mismatch. For example, thecomplementary strand of the target sequence and the targeting sequenceof the guide RNA contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. Insome embodiments, the complementary strand of the target sequence andthe targeting sequence of the guide RNA contain 1-6 mismatches. In someembodiments, the complementary strand of the target sequence and thetargeting sequence of the guide RNA contain 5 or 6 mismatches.

The length of the target sequence may depend on the nuclease systemused. For example, the target sequence for a CRISPR/Cas system comprise5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotidesin length. In some embodiments, the target sequence comprise 18-24nucleotides in length. In some embodiments, the target sequencecomprises 19-21 nucleotides in length. In some embodiments, the targetsequence comprises 20 nucleotides in length. When nickases are used, thetarget sequence comprises a pair of target sequences recognized by apair of nickases on opposite strands of the DNA molecule.

In some embodiments, the target sequence for a meganuclease comprises12-40 or more nucleotides in length. When ZFNs are used, the targetsequence comprises two half target sequences recognized by a pair ofZFNs on opposite strands of the DNA molecule, with an interconnectingsequence in between. In some embodiments, each half target sequence forZFNs independently comprise 9, 12, 15, 18, or more nucleotides inlength. In some embodiments, the interconnecting sequence for ZFNscomprise 4-20 nucleotides in length. In some embodiments, theinterconnecting sequence for ZFNs comprise 5-7 nucleotides in length.

When TALENs are used, the target sequence may similarly comprise twohalf target sequences recognized by a pair of TALENs on opposite strandsof the DNA molecule, with an interconnecting sequence in between. Insome embodiments, each half target sequence for TALENs may independentlycomprise 10-20 or more nucleotides in length. In some embodiments, theinterconnecting sequence for TALENs may comprise 4-20 nucleotides inlength. In some embodiments, the interconnecting sequence for TALENs maycomprise 12-19 nucleotides in length.

The target nucleic acid molecule is any DNA molecule that is endogenousor exogenous to a cell. As used herein, the term “endogenous sequence”refers to a sequence that is native to the cell. In some embodiments,the target nucleic acid molecule is a genomic DNA (gDNA) molecule or achromosome from a cell or in the cell. In some embodiments, the targetsequence of the target nucleic acid molecule is a genomic sequence froma cell or in the cell. In other embodiments, the cell is a eukaryoticcell. In some embodiments, the eukaryotic cell is a mammalian cell. Insome embodiments, the eukaryotic cell may be a rodent cell. In someembodiments, the eukaryotic cell may be a human cell. In furtherembodiments, the target sequence may be a viral sequence. In yet otherembodiments, the target sequence may be a synthesized sequence. In someembodiments, the target sequence may be on a eukaryotic chromosome, suchas a human chromosome.

In some embodiments, the target sequence may be located in a codingsequence of a gene, an intron sequence of a gene, a transcriptionalcontrol sequence of a gene, a translational control sequence of a gene,or a non-coding sequence between genes. In some embodiments, the genemay be a protein coding gene. In other embodiments, the gene may be anon-coding RNA gene. In some embodiments, the target sequence maycomprise all or a portion of a disease-associated gene.

In some embodiments, the target sequence may be located in a non-genicfunctional site in the genome that controls aspects of chromatinorganization, such as a scaffold site or locus control region. In someembodiments, the target sequence may be a genetic safe harbor site,i.e., a locus that facilitates safe genetic modification.

In some embodiments, the target sequence may be adjacent to aprotospacer adjacent motif (PAM), a short sequence recognized by aCRISPR/Cas9 complex. In some embodiments, the PAM may be adjacent to orwithin 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence.In some embodiments, the target sequence may include the PAM. The lengthand the sequence of the PAM may depend on the Cas9 protein used. Forexample, the PAM may be selected from a consensus or a particular PAMsequence for a specific Cas9 nuclease or Cas9 ortholog, including thosedisclosed in FIG. 1 of Ran et al., (2015) Nature, 520:186-191 (2015),which is incorporated herein by reference. In some embodiments, the PAMmay comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.Non-limiting exemplary PAM sequences include NGG (SpCas9 WT, SpCas9nickase, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9 K855A, eSpCas9 (1.0),eSpCas9 (1.1)), NGAN or NGNG (SpCas9 VQR variant), NGAG (SpCas9 EQRvariant), NGCG (SpCas9 VRER variant), NAAG (SpCas9 QQR1 variant), NNGRRTor NNGRRN (SaCas9), NNNRRT (KKH SaCas9), NNNNRYAC (CjCas9), NNAGAAW(St1Cas9), NAAAAC (TdCas9), NGGNG (St3Cas9), NG (FnCas9), NAAAAN(TdCas9), NNAAAAW (StCas9), NNNNACA (CjCas9), GNNNCNNA (PmCas9), andNNNNGATT (NmCas9) (see e.g., Cong et al., (2013) Science 339:819-823;Kleinstiver et al., (2015) Nat Biotechnol 33:1293-1298; Kleinstiver etal., (2015) Nature 523:481-485; Kleinstiver et al., (2016) Nature529:490-495; Tsai et al., (2014) Nat Biotechnol 32:569-576; Slaymaker etal., (2016) Science 351:84-88; Anders et al., (2016) Mol Cell61:895-902; Kim et al., (2017) Nat Comm 8:14500; Fonfara et al., (2013)Nucleic Acids Res 42:2577-2590; Garneau et al., (2010) Nature 468:67-71;Magadan et al., (2012) PLoS ONE 7:e40913; Esvelt et al., (2013) NatMethods 10(11):1116-1121 (wherein N is defined as any nucleotide, W isdefined as either A or T, R is defined as a purine (A) or (G), and Y isdefined as a pyrimidine (C) or (T)). In some embodiments, the PAMsequence is NGG. In some embodiments, the PAM sequence is NGAN. In someembodiments, the PAM sequence is NGNG. In some embodiments, the PAM isNNGRRT. In some embodiments, the PAM sequence is NGGNG. In someembodiments, the PAM sequence may be NNAAAAW.

Modified Donor Polynucleotides

In some embodiments, donor polynucleotides are provided with chemistriessuitable for delivery and stability within cells. Furthermore, in someembodiments, chemistries are provided that are useful for controllingthe pharmacokinetics, biodistribution, bioavailability and/or efficacyof the donor polynucleotides described herein. Accordingly, in someembodiments, donor polynucleotides described herein may be modified,e.g., comprise a modified sugar moiety, a modified internucleosidelinkage, a modified nucleoside, a modified nucleotide and/orcombinations thereof. In addition, the modified donor polynucleotidesmay exhibit one or more of the following properties: are not immunestimulatory; are nuclease resistant; have improved cell uptake comparedto unmodified donor polynucleotides; and/or are not toxic to cells ormammals.

Nucleotide and nucleoside modifications have been shown to make apolynucleotide (e.g., a donor polynucleotide) into which they areincorporated more resistant to nuclease digestion than the nativepolynucleotide and these modified polynucleotides have been shown tosurvive intact for a longer time than unmodified polynucleotides.Specific examples of modified oligonucleotides include those comprisingmodified backbones (i.e. modified internucleoside linkage), for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. In some embodiments, oligonucleotidesmay have phosphorothioate backbones; heteroatom backbones, such asmethylene(methylimino) or MMI backbones; amide backbones (see e.g., DeMesmaeker et al., Ace. Chem. Res. 1995, 28:366-374); morpholinobackbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); orpeptide nucleic acid (PNA) backbones (wherein the phosphodiesterbackbone of the polynucleotide is replaced with a polyamide backbone,the nucleotides being bound directly or indirectly to the aza nitrogenatoms of the polyamide backbone, see Nielsen et al., Science 1991, 254,1497). Phosphorus-containing modified linkages include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5031272.1 U.S. Pat. Nos. 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991. In some embodiments, the morpholino-basedoligomeric compound is a phosphorodiamidate morpholino oligomer (PMO)(e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001;and Wang et al., J. Gene Med., 12:354-364, 2010).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc, 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

In some embodiments, the donor polynucleotides of the disclosure arestabilized against nucleolytic degradation such as by the incorporationof a modification (e.g., a nucleotide modification). In someembodiments, donor polynucleotides of the disclosure include aphosphorothioate at least the first, second, and/or thirdinternucleotide linkage at the 5′ and/or 3′ end of the nucleotidesequence. In some embodiments, donor polynucleotides of the disclosureinclude one or more 2′-modified nucleotides, e.g., 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA). In some embodiments, donorpolynucleotides of the disclosure include a phosphorothioate and a2′-modified nucleotide as described herein.

Any of the modified chemistries described herein can be combined witheach other, and that one, two, three, four, five, or more differenttypes of modifications can be included within the same molecule. In someembodiments, the donor polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or modifications.

mRNA Components

In some embodiments, the systems provided by the disclosure comprise anengineered nuclease encoded by an mRNA. In some embodiments, thecompositions provided by the disclosure comprise a nuclease system,wherein the nuclease comprising the nuclease system is encoded by anmRNA. In some embodiments, the mRNA may be a naturally or non-naturallyoccurring mRNA. In some embodiments, the mRNA may include one or moremodified nucleobases, nucleosides, or nucleotides, as described below,in which case it may be referred to as a “modified mRNA”. In someembodiments, the mRNA may include a 5′ untranslated region (5′-UTR), a3′ untranslated region (3′-UTR), and/or a coding region (e.g., an openreading frame). An mRNA may include any suitable number of base pairs,including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100),hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands(e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) ofbase pairs. Any number (e.g., all, some, or none) of nucleobases,nucleosides, or nucleotides may be an analog of a canonical species,substituted, modified, or otherwise non-naturally occurring. In certainembodiments, all of a particular nucleobase type may be modified. Insome embodiments, an mRNA as described herein may include a 5′ capstructure, a chain terminating nucleotide, optionally a Kozak orKozak-like sequence (also known as a Kozak consensus sequence), astem-loop, a polyA sequence, and/or a polyadenylation signal.

A 5′ cap structure or cap species is a compound including two nucleosidemoieties joined by a linker and may be selected from a naturallyoccurring cap, a non-naturally occurring cap or cap analog, or ananti-reverse cap analog (ARCA). A cap species may include one or moremodified nucleosides and/or linker moieties. For example, a natural mRNAcap may include a guanine nucleotide and a guanine (G) nucleotidemethylated at the 7 position joined by a triphosphate linkage at their5′ positions, e.g., m⁷G(5′)ppp(5′)G, commonly written as m⁷GpppG. A capspecies may also be an anti-reverse cap analog. A non-limiting list ofpossible cap species includes m⁷GpppG, m⁷Gpppm⁷G, m⁷3′dGpppG, m₂^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, m₂ ^(7,O2′)GppppG, m⁷Gpppm⁷G,m⁷3′dGpppG, m₂ ^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, and m₂ ^(7,O2′)GppppG.

An mRNA may instead or additionally include a chain terminatingnucleoside. For example, a chain terminating nucleoside may includethose nucleosides deoxygenated at the 2′ and/or 3′ positions of theirsugar group. Such species may include 3′-deoxyadenosine (cordycepin),3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine,and 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and2′,3′-dideoxythymine. In some embodiments, incorporation of a chainterminating nucleotide into an mRNA, for example at the 3′-terminus, mayresult in stabilization of the mRNA, as described, for example, inInternational Patent Publication No. WO 2013/103659.

An mRNA may instead or additionally include a stem loop, such as ahistone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or morenucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7,or 8 nucleotide base pairs. A stem loop may be located in any region ofan mRNA. For example, a stem loop may be located in, before, or after anuntranslated region (a 5′ untranslated region or a 3′ untranslatedregion), a coding region, or a polyA sequence or tail. In someembodiments, a stem loop may affect one or more function(s) of an mRNA,such as initiation of translation, translation efficiency, and/ortranscriptional termination.

An mRNA may instead or additionally include a polyA sequence and/orpolyadenylation signal. A polyA sequence may be comprised entirely ormostly of adenine nucleotides or analogs or derivatives thereof. A polyAsequence may be a tail located adjacent to a 3′ untranslated region ofan mRNA. In some embodiments, a polyA sequence may affect the nuclearexport, translation, and/or stability of an mRNA.

Modified RNA

In some embodiments, an RNA of the disclosure (e.g.: gRNA or mRNA)comprises one or more modified nucleobases, nucleosides, nucleotides orinternucleoside linkages. In some embodiments, modified mRNAs and/orgRNAs may have useful properties, including enhanced stability,intracellular retention, enhanced translation, and/or the lack of asubstantial induction of the innate immune response of a cell into whichthe mRNA and/or gRNA is introduced, as compared to a referenceunmodified mRNA and/or gRNA. Therefore, use of modified mRNAs and/orgRNAs may enhance the efficiency of protein production, intracellularretention of nucleic acids, as well as possess reduced immunogenicity.

In some embodiments, an mRNA and/or gRNA includes one or more (e.g., 1,2, 3 or 4) different modified nucleobases, nucleosides, nucleotides orinternucleoside linkages. In some embodiments, an mRNA and/or gRNAincludes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or more) different modified nucleobases,nucleosides, or nucleotides. In some embodiments, the modified gRNA mayhave reduced degradation in a cell into which the gRNA is introduced,relative to a corresponding unmodified gRNA. In some embodiments, themodified mRNA may have reduced degradation in a cell into which the mRNAis introduced, relative to a corresponding unmodified mRNA.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-0H-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includeα-thio-adenosine, 2-amino-purine, 2,6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-0H-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeα-thio-guanosine, inosine (I), 1-methyl-inosine (m¹I), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OhyW),undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine(m² ₂G), N2,7-dimethyl-guanosine (m²′⁷G), N2, N2,7-dimethyl-guanosine(m^(2,2,7)G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine,N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine(Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In some embodiments, an mRNA and/or gRNA of the disclosure includes acombination of one or more of the aforementioned modified nucleobases(e.g., a combination of 2, 3 or 4 of the aforementioned modifiednucleobases.)

In some embodiments, the modified nucleobase is pseudouridine (w),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.) In one embodiment, themodified nucleobase is N1-methylpseudouridine (m¹ψ) and the mRNA of thedisclosure is fully modified with N1-methylpseudouridine (m¹ψ). In someembodiments, N1-methylpseudouridine (m¹ψ) represents from 75-100% of theuracils in the mRNA. In some embodiments, N1-methylpseudouridine (m¹ψ)represents 100% of the uracils in the mRNA.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s²C),2-thio-5-methyl-cytidine. In some embodiments, an mRNA of the disclosureincludes a combination of one or more of the aforementioned modifiednucleobases (e.g., a combination of 2, 3 or 4 of the aforementionedmodified nucleobases.)

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A),N6-methyl-adenosine (m⁶A). In some embodiments, an mRNA of thedisclosure includes a combination of one or more of the aforementionedmodified nucleobases (e.g., a combination of 2, 3 or 4 of theaforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), 7-methyl-guanosine (m⁷G),1-methyl-guanosine (m¹G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. Insome embodiments, an mRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In certain embodiments, an mRNA and/or a gRNA of the disclosure isuniformly modified (i.e., fully modified, modified through-out theentire sequence) for a particular modification. For example, an mRNA canbe uniformly modified with N1-methylpseudouridine (m¹ψ) or5-methyl-cytidine (m⁵C), meaning that all uridines or all cytosinenucleosides in the mRNA sequence are replaced withN1-methylpseudouridine (m¹ψ) or 5-methyl-cytidine (m⁵C). Similarly,mRNAs of the disclosure can be uniformly modified for any type ofnucleoside residue present in the sequence by replacement with amodified residue such as those set forth above.

In some embodiments, an mRNA of the disclosure may be modified in acoding region (e.g., an open reading frame encoding a polypeptide). Inother embodiments, an mRNA may be modified in regions besides a codingregion. For example, in some embodiments, a 5′-UTR and/or a 3′-UTR areprovided, wherein either or both may independently contain one or moredifferent nucleoside modifications. In such embodiments, nucleosidemodifications may also be present in the coding region.

Ribonucleoproteins

In certain aspects, the site-directed polypeptide (e.g., Cas nuclease)and genome-targeting nucleic acid (e.g., gRNA or sgRNA) may each beadministered separately to a cell or a subject. In certain aspects, thesite-directed polypeptide may be pre-complexed with one or more guideRNAs, or one or more sgRNAs. Such pre-complexed material is known as aribonucleoprotein particle (RNP). In some embodiments, the nucleasesystem comprises a ribonucleoprotein (RNP). In some embodiments, thenuclease system comprises a Cas9 RNP comprising a purified Cas9 proteinin complex with a gRNA. Cas9 protein can be expressed and purified byany means known in the art. Ribonucleoproteins are assembled in vitroand can be delivered directly to cells using standard electroporation ortransfection techniques known in the art.

Vectors

In some embodiments, the site-directed nuclease (e.g., Cas nuclease) andthe donor polynucleotide may be provided by one or more vectors. In someembodiments, the vector may be a DNA vector. In some embodiments, thevector may be circular. In other embodiments, the vector may be linear.Non-limiting exemplary vectors include plasmids, phagemids, cosmids,artificial chromosomes, minichromosomes, transposons, viral vectors, andexpression vectors.

In some embodiments, the vector may be a viral vector. In someembodiments, the viral vector may be genetically modified from itswild-type counterpart. For example, the viral vector may comprise aninsertion, deletion, or substitution of one or more nucleotides tofacilitate cloning or such that one or more properties of the vector ischanged. Such properties may include packaging capacity, transductionefficiency, immunogenicity, genome integration, replication,transcription, and translation. In some embodiments, a portion of theviral genome may be deleted such that the virus is capable of packagingexogenous sequences having a larger size. In some embodiments, the viralvector may have an enhanced transduction efficiency. In someembodiments, the immune response induced by the virus in a host may bereduced. In some embodiments, viral genes (such as, e.g., integrase)that promote integration of the viral sequence into a host genome may bemutated such that the virus becomes non-integrating. In someembodiments, the viral vector may be replication defective. In someembodiments, the viral vector may comprise exogenous transcriptional ortranslational control sequences to drive expression of coding sequenceson the vector. In some embodiments, the virus may be helper-dependent.For example, the virus may need one or more helper virus to supply viralcomponents (such as, e.g., viral proteins) required to amplify andpackage the vectors into viral particles. In such a case, one or morehelper components, including one or more vectors encoding the viralcomponents, may be introduced into a host cell along with the vectorsystem described herein. In other embodiments, the virus may behelper-free. For example, the virus may be capable of amplifying andpackaging the vectors without any helper virus. In some embodiments, thevector system described herein may also encode the viral componentsrequired for virus amplification and packaging.

Non-limiting exemplary viral vectors include adeno-associated virus(AAV) vector, lentivirus vectors, adenovirus vectors, herpes simplexvirus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, andretrovirus vectors. In some embodiments, the viral vector may be an AAVvector. In other embodiments, the viral vector may a lentivirus vector.In some embodiments, the lentivirus may be non-integrating. In someembodiments, the viral vector may be an adenovirus vector. In someembodiments, the adenovirus may be a high-cloning capacity or “gutless”adenovirus, where all coding viral regions apart from the 5′ and 3′inverted terminal repeats (ITRs) and the packaging signal (Ψ) aredeleted from the virus to increase its packaging capacity. In yet otherembodiments, the viral vector may be an HSV-1 vector. In someembodiments, the HSV-1-based vector is helper dependent, and in otherembodiments it is helper independent. For example, an amplicon vectorthat retains only the packaging sequence requires a helper virus withstructural components for packaging, while a 30 kb-deleted HSV-1 vectorthat removes non-essential viral functions does not require helpervirus. In additional embodiments, the viral vector may be bacteriophageT4. In some embodiments, the bacteriophage T4 may be able to package anylinear or circular DNA or RNA molecules when the head of the virus isemptied. In further embodiments, the viral vector may be a baculovirusvector. In yet further embodiments, the viral vector may be a retrovirusvector. In embodiments using AAV or lentiviral vectors, which havesmaller cloning capacity, it may be necessary to use more than onevector to deliver all the components of a vector system as disclosedherein. For example, one AAV vector may contain sequences encoding aCas9 protein, while a second AAV vector may contain one or more guidesequences and one or more copies of donor polynucleotide.

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12,AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in,for example, international patent application publication number WO01/83692. In some embodiments, the vector is AAV6.

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such asaneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line can then be infected with ahelper virus, such as adenovirus. The advantages of this method are thatthe cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirusorbaculovirus, rather than plasmids, to introduce rAAV genomes and/orrep and cap genes into packaging cells.

In certain embodiments, a viral vector may be modified to target aparticular tissue or cell type. For example, viral surface proteins maybe altered to decrease or eliminate viral protein binding to its naturalcell surface receptor(s). The surface proteins may also be engineered tointeract with a receptor specific to a desired cell type. Viral vectorsmay have altered host tropism, including limited or redirected tropism.Certain engineered viral vectors are described, for example, inWO2011130749, WO2015009952, U.S. Pat. No. 5,817,491, WO2014135998, andWO2011125054. In some embodiments, the vector may be capable of drivingexpression of one or more coding sequences in a cell. In someembodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast,plant, insect, or mammalian cell. In some embodiments, the eukaryoticcell may be a mammalian cell. In some embodiments, the eukaryotic cellmay be a rodent cell. In some embodiments, the eukaryotic cell may be ahuman cell. Suitable promoters to drive expression in different types ofcells are known in the art. In some embodiments, the promoter may bewild-type. In other embodiments, the promoter may be modified for moreefficient or efficacious expression. In yet other embodiments, thepromoter may be truncated yet retain its function. For example, thepromoter may have a normal size or a reduced size that is suitable forproper packaging of the vector into a virus.

In some embodiments, the vector may comprise a nucleotide sequenceencoding the nuclease described herein. In some embodiments, the vectorsystem may comprise one copy of the nucleotide sequence encoding thenuclease. In other embodiments, the vector system may comprise more thanone copy of the nucleotide sequence encoding the nuclease. In someembodiments, the nucleotide sequence encoding the nuclease may beoperably linked to at least one transcriptional or translational controlsequence. In some embodiments, the nucleotide sequence encoding thenuclease may be operably linked to at least one promoter. In someembodiments, the nucleotide sequence encoding the nuclease may beoperably linked to at least one transcriptional or translational controlsequence.

In some embodiments, the promoter may be constitutive, inducible, ortissue-specific. In some embodiments, the promoter may be a constitutivepromoter. Non-limiting exemplary constitutive promoters includecytomegalovirus immediate early promoter (CMV), simian virus (SV40)promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV)promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglyceratekinase (PGK) promoter, elongation factor-alpha (EF1α) promoter,ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulinpromoters, a functional fragment thereof, or a combination of any of theforegoing. In some embodiments, the promoter may be a CMV promoter. Insome embodiments, the promoter may be a truncated CMV promoter. In otherembodiments, the promoter may be an EF1α promoter. In some embodiments,the promoter may be an inducible promoter. Non-limiting exemplaryinducible promoters include those inducible by heat shock, light,chemicals, peptides, metals, steroids, antibiotics, or alcohol. In someembodiments, the inducible promoter may be one that has a low basal(non-induced) expression level, such as, e.g., the Tet-On® promoter(Clontech). In some embodiments, the promoter may be a tissue-specificpromoter. In some embodiments, the tissue-specific promoter isexclusively or predominantly expressed in liver tissue. Non-limitingexemplary tissue-specific promoters include B29 promoter, CD14 promoter,CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAPpromoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter,Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASPpromoter.

In some embodiments, the nuclease encoded by the vector may be a Casprotein, such as a Cas9 protein or Cpf1 protein. The vector system mayfurther comprise a vector comprising a nucleotide sequence encoding theguide RNA described herein. In some embodiments, the vector system maycomprise one copy of the guide RNA. In other embodiments, the vectorsystem may comprise more than one copy of the guide RNA. In embodimentswith more than one guide RNA, the guide RNAs may be non-identical suchthat they target different target sequences, or have other differentproperties, such as activity or stability within the Cas9 RNP complex.In some embodiments, the nucleotide sequence encoding the guide RNA maybe operably linked to at least one transcriptional or translationalcontrol sequence. In some embodiments, the nucleotide sequence encodingthe guide RNA may be operably linked to at least one promoter. In someembodiments, the promoter may be recognized by RNA polymerase III (PolIII). Non-limiting examples of Pol III promoters include U6, H1 and tRNApromoters. In some embodiments, the nucleotide sequence encoding theguide RNA may be operably linked to a mouse or human U6 promoter. Inother embodiments, the nucleotide sequence encoding the guide RNA may beoperably linked to a mouse or human H1 promoter. In some embodiments,the nucleotide sequence encoding the guide RNA may be operably linked toa mouse or human tRNA promoter. In embodiments with more than one guideRNA, the promoters used to drive expression may be the same ordifferent. In some embodiments, the nucleotide encoding the crRNA of theguide RNA and the nucleotide encoding the tracr RNA of the guide RNA maybe provided on the same vector. In some embodiments, the nucleotideencoding the crRNA and the nucleotide encoding the tracr RNA may bedriven by the same promoter. In some embodiments, the crRNA and tracrRNA may be transcribed into a single transcript. For example, the crRNAand tracr RNA may be processed from the single transcript to form adouble-molecule guide RNA. Alternatively, the crRNA and tracr RNA may betranscribed into a single-molecule guide RNA. In other embodiments, thecrRNA and the tracr RNA may be driven by their corresponding promoterson the same vector. In yet other embodiments, the crRNA and the tracrRNA may be encoded by different vectors.

In some embodiments, the nucleotide sequence encoding the guide RNA maybe located on the same vector comprising the nucleotide sequenceencoding a Cas9 protein. In some embodiments, expression of the guideRNA and of the Cas9 protein may be driven by different promoters. Insome embodiments, expression of the guide RNA may be driven by the samepromoter that drives expression of the Cas9 protein. In someembodiments, the guide RNA and the Cas9 protein transcript may becontained within a single transcript. For example, the guide RNA may bewithin an untranslated region (UTR) of the Cas9 protein transcript. Insome embodiments, the guide RNA may be within the 5′ UTR of the Cas9protein transcript. In other embodiments, the guide RNA may be withinthe 3′ UTR of the Cas9 protein transcript. In some embodiments, theintracellular half-life of the Cas9 protein transcript may be reduced bycontaining the guide RNA within its 3′ UTR and thereby shortening thelength of its 3′ UTR. In additional embodiments, the guide RNA may bewithin an intron of the Cas9 protein transcript. In some embodiments,suitable splice sites may be added at the intron within which the guideRNA is located such that the guide RNA is properly spliced out of thetranscript. In some embodiments, expression of the Cas9 protein and theguide RNA in close proximity on the same vector may facilitate moreefficient formation of the CRISPR complex.

In some embodiments, the vector system may further comprise a vectorcomprising the donor polynucleotide described herein. In someembodiments, the vector system may comprise one copy of the donorpolynucleotide. In other embodiments, the vector system may comprisemore than one copy of the donor polynucleotide. In some embodiments, thevector system may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies ofthe donor polynucleotide. The multiple copies of the donorpolynucleotide may be located on the same or different vectors. Themultiple copies of the donor polynucleotide may also be adjacent to oneanother, or separated by other nucleotide sequences or vector elements.

A vector system may comprise 1-3 vectors. In some embodiments, thevector system may comprise one single vector. In other embodiments, thevector system may comprise two vectors. In additional embodiments, thevector system may comprise three vectors. When different guide RNAs ordonor polynucleotides are used for multiplexing, or when multiple copiesof the guide RNA or the donor polynucleotide are used, the vector systemmay comprise more than three vectors.

In some embodiments, the nucleotide sequence encoding a Cas9 protein, anucleotide sequence encoding the guide RNA, and a donor polynucleotidemay be located on the same or separate vectors. In some embodiments, allof the sequences may be located on the same vector. In some embodiments,two or more sequences may be located on the same vector. The sequencesmay be oriented in the same or different directions and in any order onthe vector. In some embodiments, the nucleotide sequence encoding theCas9 protein and the nucleotide sequence encoding the guide RNA may belocated on the same vector. In some embodiments, the nucleotide sequenceencoding the Cas9 protein and the donor polynucleotide may be located onthe same vector. In some embodiments, the nucleotide sequence encodingthe guide RNA and the donor polynucleotide may be located on the samevector. In some embodiments, the vector system may comprise a firstvector comprising the nucleotide sequence encoding the Cas9 protein, anda second vector comprising the nucleotide sequence encoding the guideRNA and the donor polynucleotide.

Methods of Increasing Homology Directed Repair

The repair of DNA breaks (e.g., DSBs) in cells is accomplished primarilythrough two DNA repair pathways, namely the non-homologous end joining(NHEJ) repair pathway and homology-directed repair (HDR) pathway.

During NHEJ, the Ku70/80 heterodimers bind to DNA ends and recruit theDNA protein kinase (DNA-PK) (Cannan & Pederson (2015) J Cell Physiol231:3-14). Once bound, DNA-PK activates its own catalytic subunit(DNA-PKcs) and further enlists the endonuclease Artemis (also known asSNM1c). At a subset of DSBs, Artemis removes excess single-strand DNA(ssDNA) and generates a substrate that will be ligated by DNA ligase IV.DNA repair by NHEJ involves blunt-end ligation mechanism independent ofsequence homology via the canonical DNA-PKcs/Ku70/80 complex.

During DNA repair by HDR, DSB ends are resected to expose 3′ ssDNAtails, primarily by the MRE11-RAD50-NBS1 (MRN) complex (Heyer et al.,(2010) Annu Rev Genet 44: 113-139). Under physiological conditions, theadjacent sister chromatid will be used as a repair template, providing ahomologous sequence, and the ssDNA will invade the template mediated bythe recombinase Rad51, displacing an intact strand to form a D-loop.D-loop extension is followed by branch migration to producedouble-Holliday junctions, the resolution of which completes the repaircycle. HDR often requires error-prone polymerases yet is typicallyviewed as error-free (Li and Xu (2016) Acta Biochim Biophys Sin48(7):641-646).

The NHEJ pathway limits HDR first by being a fast-acting repair pathwaythat seals the broken DNA ends through a DNA ligase IV-dependentmechanism. Secondly, in NHEJ the Ku70/Ku80 heterodimer binds to the DNAends with high affinity to block their processing by the nucleases thatgenerate the single-stranded DNA tails that are necessary for initiationof HDR (Lieber, M. et al. (2010) Annu Rev Biochem 79:181-211; Symington,L. et al. (2011) Annu Review Genetics 45:247-271). Thirdly, 53BP1 isactively recruited to sites of damaged chromatin present at a DNA DSBwhere it functions to suppress the formation of 3′ ssDNA tails andantagonize the action of BRCA1, a factor involved in HDR(Escribano-Diaz, C. (2013) Molecular cell 49:872-883; Feng, L. et al.(2013) J. Biol Chem. 288:11135-11143).

During the cell cycle, NHEJ occurs predominantly during G0/G1 and G2(Chiruvella et al., (2013) Cold Spring Harb Perspect Biol 5:a012757).Current studies have shown that NHEJ is the only DSB repair pathwayactive during G0 and G1, while HDR functions primarily during the S andG2 phases, playing a major role in the repair of replication-associatedDSBs (Karanam et al., (2012) Mol Cell 47:320-329; Li and Xu (2016) ActaBiochim Biophys Sin 48(7):641-646). NHEJ, unlike HDR, is active in bothdividing and non-dividing cells, not just dividing cells, which enablesthe development of therapies based on genome editing for non-dividingadult cells, such as, for example, cells of the eye, brain, pancreas, orheart.

A third repair mechanism is microhomology-mediated end joining (MMEJ),also referred to as “Alternative NHEJ”, in which the genetic outcome issimilar to NHEJ in that small deletions and insertions can occur at thecleavage site. MMEJ makes use of homologous sequences of a fewnucleotides flanking the DNA break site to drive a more favored DNA endjoining repair outcome, and recent reports have further elucidated themolecular mechanism of this process (Cho and Greenberg, (2015) Nature518:174-176; Mateos-Gomez et al., (2015) Nature 518, 254-257; Ceccaldiet al., (2015) Nature 528, 258-262). The key mechanistic steps areresection of DSB ends, annealing of microhomologous regions, removal ofheterologous flaps, fill-in synthesis and ligation. PARP1 plays a keyrole in binding to DNA blunt ends and initiating the MMEJ pathway byrecruiting DNA polymerase theta (Polio). Pol0 enables the formation ofresected DNA ends, as well as enabling the fill-in synthesis (Wang. H.et al. (2017) Cell Biosci 7:6).

Inhibition of 53BP1

In some embodiments, the disclosure provides methods for increasing HDRof a DSB mediated by a site-directed nuclease in a target gene in a cellor population of cells, such as a quiescent cell that has been inducedto divide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by inhibition of 53BP1. In some embodiments,the disclosure provides methods for increasing HDR of a DSB mediated bya site-directed nuclease in a cell or population of cells expressing anE6V mutation in HBB, by inhibition of 53BP1.

The p53-binding protein 1 (53BP1) is a key regulator of cellularresponse to DNA damage. The choice of repair pathway for repair of a DNADSB is largely controlled by an antagonism between 53BP1, a pro-NHEJfactor, and BRCA1, a pro-HDR factor (Chapman, J. et al. (2012) Molecularcell 47:497-510). 53BP1 promotes NHEJ repair over HDR repair bysuppressing formation of 3′ single-stranded DNA tails, which is therate-limiting step in the initiation of the HDR pathway, and byinhibiting BRCA1 recruitment to DSB sites (Escribano-Diaz, C. et al.(2013) Mol Cell. 49:872-883; Feng, L. et al (2013) J Biol Chem288:11135-11143). Loss of 53BP1 has been shown to increase HDRefficiency, (Canny, M. et al. (2018) Nat Biotechnol. 36(1):95-102).Thus, inhibition of 53BP1 is expected to reduce DSB repair by the NHEJpathway and favor repair by the HDR pathway.

Distinct protein domains in the 53BP1 structure are required to enableits function as a pro-NHEJ factor (Zimmermann et al (2014) Trends CellBiol 24:108-117). Human 53BP1 is a large (e.g., 200 kDa, 1972 aminoacids) multi-domain protein that enables recruitment to DSB sites andbinding of protein factors involved in DNA repair. The 53BP1 N-terminusis comprised of a large subunit that is heavily phosphorylated followingDNA damage and facilitates binding interactions with DNA repairmachinery. The central portion of 53BP1 comprises a focus-forming regionthat is essential for binding to damaged chromatin, which allowsrecruitment to DSB sites. It comprises a nuclear localization signal(NLS), a tandem Tudor domain that binds to di-methylated histone H4lysine 20 (e.g., H4K20^(Me2)), and a ubiquitin-dependent recruitment(UDR) motif that recognizes histone H2A/H2AX ubquitinated on lysine 15(e.g., H2A(X)K15^(Ub)) (Botuyan, M. (2006) Cell 127:1361-1373;Fradet-Turcotte et al (2013) Nature 499:50-54). The focus-forming regionextends from amino acids 1220-1711 of human 53BP1, with the tandem Tudordomain extending from amino acids 1484-1603 and the UDR extending fromamino acids 1604-1631. The 53BP1 C-terminus is comprised of repeatingBRCA1 C-terminus (BRCT) domains that are important for DNA repair inheterochromatin (Noon et al (2010) Nat Cell Biol 12:177-184) and mediateinteractions with the tumor suppressor p53 that guides cellular responseto DNA damage (Iwabuchi, et al (1994) PNAS 91:6098-6102).

The functionality of 53BP1 for promoting the NHEJ pathway requiresrecruitment to damaged chromatin through its tandem Tudor and UDRdomains and binding to repair machinery through phosphorylation of the53BP1 N-terminus.

Accordingly, the present disclosure provides 53BP1 inhibitors thatinhibit NHEJ and promote HDR repair of a DSB in a target gene. In someembodiments, a 53BP1 inhibitor of the disclosure inhibits 53BP1recruitment to DSB sites. In some embodiments, a 53BP1 inhibitor of thedisclosure inhibits 53BP1 recruitment by inhibiting, reducing,disrupting or blocking an interaction of 53BP1 with damaged chromatin.In some embodiments, a 53BP1 inhibitor of the disclosure inhibits,reduces, disrupts or blocks an interaction of the 53BP1 focus formingregion (amino acids 1220-1711) with DSB sites. In some embodiments, a53BP1 inhibitor of the disclosure inhibits, reduces, disrupts or blocksan interaction of the 53BP1 focus forming region (amino acids 1220-1711)with damaged chromatin. In some embodiments, a 53BP1 inhibitor of thedisclosure inhibits, reduces, disrupts or blocks an interaction of the53BP1 tandem Tudor domain with damaged chromatin (e.g., with methylatedhistone, H4K20^(Me2)). In some embodiments, a 53BP1 inhibitor of thedisclosure inhibits, reduces, disrupts or blocks the interaction of the53BP1 UDR motif with damaged chromatin (e.g., with ubquitinylatedhistone, H2A(X)K15^(Ub)).

In some embodiments, a 53BP1 inhibitor of the disclosure inhibits,reduces, disrupts or blocks protein-protein interactions with the 53BP1BRCT domain. In some embodiments, a 53BP1 inhibitor of the disclosureinhibits, reduces, disrupts or blocks the interactions of the 53BP1 BRCTdomain with the tumor suppressor p53.

In some embodiments, a 53BP1 inhibitor of the disclosure inhibits,reduces, disrupts or blocks the ability of 53BP1 to bind to DNA repairfactors. In some embodiments, a 53BP1 inhibitor of the disclosureinhibits, reduces, disrupts or blocks phosphorylation of the 53BP1N-terminus, thus inhibiting, reducing or preventing binding of DNArepair factors. In some embodiments, a 53BP1 inhibitor of the disclosurebinds to phosphorylated sites on the 53BP1 N-terminus, thus inhibiting,reducing or preventing DNA repair factors from recognizing and bindingto phosphorylated sites on the 53BP1 N-terminus. In some embodiments, a53BP1 inhibitor of the disclosure reduces, eliminates or removesphosphorylated sites on the 53BP1 N-terminus (e.g., by promoting orcatalyzing a dephosphorylation mechanism), thus reducing, eliminating orremoving sites required for binding of DNA repair factors. In someembodiments, a 53BP1 inhibitor that binds to phosphorylated sites on53BP1 and facilitates HDR is suppressor of cancer cell invasion (SCAI)or a fragment thereof. In some embodiments, binding of SCAI or afragment thereof prevents binding of the DNA repair factorRAP1-interacting factor homolog (RIF1). In some embodiments, blockingRIF1 binding to 53BP1 results in increased HDR repair of a DNA DSB.

In some embodiments, the 53BP1 inhibitor of the disclosure inhibits,disrupts or blocks 53BP1 recruitment to DSB sites in the cell. In someembodiments, the 53BP1 inhibitor of the disclosure inhibits, disrupts orblocks an interaction of 53BP1 with damaged chromatin in the cell. Insome embodiments, the 53BP1 inhibitor of the disclosure inhibits,disrupts or blocks binding of DNA repair factors to sites ofphosphorylation on the 53BP1 N-terminus. In some embodiments, the 53BP1inhibitor of the disclosure is a small molecule. In some embodiments,the 53BP1 inhibitor of the disclosure is a polypeptide. In someembodiments, the 53BP1 inhibitor of the disclosure is a nucleic acid.

In some embodiments, recruitment of 53BP1 to a DSB site occurs viarecognition of damaged chromatin. In some embodiments, recruitment of53BP1 to damaged chromatin occurs through recognition of H4K20me2through the 53BP1 UDR motif. In some embodiments, recognition of damagedchromatin by 53BP1 is dependent upon ubiquitination of histones. In someembodiments, inhibition of histone ubiquitination results in inhibitionof 53BP1 recruitment to DSB sites.

Acetylation of 53BP1 has been shown to inhibit 53BP1 binding to damagedchromatin (Guo et al (2018) Nucleic Acids Res 46:689-703). In someembodiments, an inhibitor of 53BP1 promotes post-translationalmodification of 53BP1. In some embodiments, an inhibitor of 53BP1promotes post-translation modification of 53BP1 that prevents 53BP1binding to damaged chromatin. In some embodiments, an inhibitor of 53BP1promotes acetylation of 53BP1. In some embodiments, an inhibitor of53BP1 promotes acetylation of the 53BP1 UDR motif. In some embodiments,acetylation of 53BP1 prevents 53BP1 recruitment to DSB sites.

In some embodiments, a 53BP1 inhibitor is identified by binding affinityfor the 53BP1 polypeptide. Methods of measuring binding affinity of aninhibitor to a protein are known in the art. Non-limiting examplesinclude measuring inhibitor affinity by enzyme-linked immunosorbentassay (e.g., ELISA), immunoblot, immunoprecipitation-based assay,fluorescence polarization assay, fluorescence resonance energy transferassay, fluorescence anisotropy assay, yeast surface display (Gai (2007)Curr Opin Struct Biol 17:467-473), kinetic exclusion assay, surfaceplasmon resonance, or isothermal titration calorimetry. In someembodiments, a method of measuring binding affinity is an ELISA whereinan inhibitor is measured for affinity to the 53BP1 polypeptide. In someembodiments, binding affinity is evaluated by a competition-based ELISAwherein binding of an inhibitor to the 53BP1 polypeptide is measured inthe presence of increasing concentrations of a known 53BP1 bindingpartner (e.g., a histone methyl-lysine peptide with affinity for 53BP1).

In some embodiments, a 53BP1 inhibitor is identified by binding affinityfor a fragment of the 53BP1 polypeptide. In some embodiments, a fragmentis a domain of the 53BP1 polypeptide. In some embodiments, the domain isthe Tudor domain. In some embodiments, the domain is the UDR motif. Insome embodiments, the domain comprises the N-terminus of the 53BP1polypeptide.

In some embodiments, a 53BP1 inhibitor of the disclosure binds to the53BP1 polypeptide. Methods of determining the structural interactionsthat enable binding of the inhibitor with the 53BP1 polypeptide areknown in the art. Non-limiting examples include X-ray crystallography,nuclear magnetic resonance (NMR) spectroscopy, electron microscopy,small-angle X-ray scattering (SAXS), and small-angle neutron scattering(SANS). In some embodiments, the structural interactions are determinedby a mutagenesis experiment wherein residues of the 53BP1 polypeptideare mutated and the effect on inhibitor binding are evaluated. Suchmethods enable identification of key residues that contribute tobinding.

In some embodiments, the 53BP1 inhibitor of the disclosure is a 53BP1binding polypeptide that inhibits 53BP1 recruitment to the DSB in thecell. In some embodiments, a 53BP1 binding polypeptide of the disclosureinhibits, disrupts or blocks binding of 53BP1 to damaged chromatin inthe cell. In some embodiments, a 53BP1 binding polypeptide of thedisclosure inhibits, disrupts or blocks the 53BP1 tandem Tudor domainfrom binding to damaged chromatin in the cell. In some embodiments, a53BP1 binding polypeptide of the disclosure inhibits, disrupts or blocksthe 53BP1 UDR motif from binding to damaged chromatin in the cell.

In some embodiments, an inhibitor of 53BP1 is a polypeptide identifiedfrom a phage-display library or a variant thereof as described by US2019/0010196A, which is incorporated by reference herein. In someembodiments, a polypeptide inhibitor of 53BP1 has binding affinity forthe 53BP1 Tudor domain. The 53BP1 Tudor domain is involved inrecognition of methylated residues on the histone core that facilitatesrecruitment of 53BP1 to a DNA DSB site. In some embodiments, a 53BP1polypeptide inhibitor of the disclosure inhibits, reduces or preventsrecruitment of 53BP1 to a DNA DSB by binding to the 53BP1 Tudor domain.

In some embodiments, a 53BP1 polypeptide inhibitor of the disclosure ismodified, by, for example, substitution of one or more amino acidresidues, insertion of one or more amino acid residues, or deletion ofone or more amino acid residues. In some embodiments, a 53BP1polypeptide inhibitor of the disclosure is modified by chemicalmodifications. Techniques for modification of one or more amino acidresidues are known to one skilled in the art. In some embodiments, amodification is substitution of one or more amino acid residues. In oneembodiment, a modification increases binding affinity of the 53BP1polypeptide inhibitor for the 53BP1 polypeptide or a fragment thereof.

In some embodiments, a modified polypeptide inhibitor of 53BP1 isidentified by affinity for the 53BP1 Tudor domain. Affinity for the53BP1 Tudor domain may be assessed by suitable assays known to oneskilled in the art. In some embodiments, affinity is measured by acompetitive immunoprecipitation assay against an endogenous polypeptidethat binds 53BP1, for example, dimethylated histone H4 Lys20. In someembodiments, affinity is measured by isothermal calorimetry usingrecombinant 53BP1. In some embodiments, affinity is determined byassessing 53BP1 recruitment to DSB sites. In some embodiments, a 53BP1polypeptide inhibitor of the disclosure has a quantifiable bindingaffinity for the 53BP1 Tudor domain of approximately 0.5 to 15×10⁻⁹ M,0.5 to 25×10⁻⁹, 0.5 to 50×10⁻⁹ M, 0.5 to 100×10⁻⁹ M, 0.5 to 200×10⁻⁹ M,1 to 200×10⁻⁹ M, 1 to 300×10⁻⁹ M, 1 to 400×10⁻⁹ M, 1 to 500×10⁻⁹ M, 100to 250×10⁻⁹ M, 100 to 500×10⁻⁹ M, or 200 to 500×10⁻⁹ M. In someembodiments, a 53BP1 polypeptide inhibitor of the disclosure has aquantifiable binding affinity for the 53BP1 Tudor domain ofapproximately 200 to 500×10⁻⁹ M. In some embodiments, a 53BP1polypeptide inhibitor of the disclosure has a quantifiable bindingaffinity for the 53BP1 Tudor domain of approximately 250×10⁻⁹ M.

In some embodiments, a 53BP1 polypeptide inhibitor of the disclosurecomprises a polypeptide sequence of SEQ ID NO: 70. In some embodiments,a 53BP1 polypeptide inhibitor of the disclosure comprises a polypeptidesequence that is at least about 50%, 60%, 70% or 80% identical to thepolypeptide sequence of SEQ ID NO: 70. In some embodiments, a 53BP1polypeptide inhibitor comprises a polypeptide sequence that is at leastabout 90%, 95%, 96%, 97%, 98% or 99% identical to the polypeptidesequence of SEQ ID NO: 70. In some embodiments, a 53BP1 polypeptideinhibitor of the disclosure comprises a polypeptide sequence that is atleast about 95% identical to the polypeptide sequence of SEQ ID NO: 70.In some embodiments, a 53BP1 polypeptide inhibitor of the disclosurecomprises a polypeptide sequence that is at least about 96% identical tothe polypeptide sequence of SEQ ID NO: 70. In some embodiments, a 53BP1polypeptide inhibitor of the disclosure comprises a polypeptide sequencethat is at least about 97% identical to the polypeptide sequence of SEQID NO: 70. In some embodiments, a 53BP1 polypeptide inhibitor of thedisclosure comprises a polypeptide sequence that is at least about 98%identical to the polypeptide sequence of SEQ ID NO: 70. In someembodiments, a 53BP1 polypeptide inhibitor of the disclosure comprises apolypeptide sequence that is at least about 99% identical to thepolypeptide sequence of SEQ ID NO: 70. In some embodiments, percentidentity is made by a comparison that is performed by a BLAST algorithmwherein the parameters of the algorithm are selected to encompass thelargest match between the respective polypeptide sequences over theentire length of the polypeptide sequence as set forth by SEQ ID NO: 70.BLAST algorithms are often used for sequence analysis and are well knownby one skilled in the art (Altschul, S., et al. (1990) J Mol. Biol215:403-410; Gish, W. et al. (1993) Nat. Genet. 3:266-272; Madden, T. etal. (1996) Meth. Enzymol. 266:131-141; Altschul, S. et al. (1997)Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res.7:649-656; Wootton, J. et al., (1993) Comput. Chem. 17:149-163; Hancock,J. et al. (1994) Comput. Appl. Biosci. 10:67-70).

In some embodiments, a 53BP1 polypeptide inhibitor of the disclosurecomprises a fragment of a polypeptide comprising the polypeptidesequence of SEQ ID NO: 70 that retains binding to the 53BP1 Tudordomain. In some embodiments, a fragment has at least 1-5, at least 1-10,at least 5-15, at least 10-20, at least 15-30, at least 15-40 feweramino acid residues than a polypeptide comprising a polypeptide sequenceas set forth by SEQ ID NO: 70.

In some embodiments, a 53BP1 polypeptide inhibitor of the disclosurecomprises a fusion polypeptide comprising a polypeptide comprising thepolypeptide sequence of SEQ ID NO: 70 that retains binding to the 53BP1Tudor domain. In some embodiments, a fusion polypeptide is obtained byaddition of amino acids or peptides or by substitutions of individualamino acids or peptides that enable by chemical coupling with suitablereagents to a fusion partner. In some embodiments, a fusion is preparedby preparation and expression of a vector comprising a gene encoding apolypeptide described herein and a gene encoding a fusion partner. Insome embodiments, a fusion partner is a polypeptide, non-limitingexamples include an enzyme, a fluorescent tag, a purification tag, atoxin, an antibody fragment, or an albumin fragment. In someembodiments, a fusion partner is a chemical label, non-limiting examplesinclude a fluorescent dye, biotin, a radioactive label, a saccharide, ora phosphate.

In some embodiments, a 53BP1 polypeptide inhibitor as described hereinis encoded by a polynucleotide. In some embodiments, a 53BP1 polypeptideinhibitor as described herein is provided as a nucleic acid comprising anucleotide sequence encoding the 53BP1 polypeptide inhibitor. In someembodiments, the nucleic acid is a DNA molecule. In some embodiments,the nucleic acid is an RNA molecule. In some embodiments, the nucleicacid is a messenger RNA (mRNA). Methods of preparing mRNA or highexpression of an encoded polypeptide are known in the art. In someembodiments, an mRNA comprises an open-reading frame (ORF) encoding aninhibitor of 53BP1. In some embodiments, the nucleic acid encoding a53BP1 polypeptide inhibitor comprises an mRNA comprising an ORF encodingthe amino acid sequence of SEQ ID NO: 70.

In some embodiments, a nucleic acid comprising a nucleotide sequenceencoding a 53BP1 polypeptide inhibitor is delivered to a cell by avector. Methods of delivering nucleic acids to a cell using a vector areknown in the art and are described herein.

In some embodiments, a 53BP1 inhibitor of the disclosure comprises agene-editing system for disrupting a gene encoding 53BP1. In someembodiments, the 53BP1 inhibitor comprises a CRISPR/Cas9 gene editingsystem. Methods of using CRISPR-Cas gene editing technology to create agenomic deletion in a cell (e.g., a knock-out in a gene of a cell) areknown (e.g., Bauer (2015) Vis Exp 95:e52118). In some embodiments, aknock-out of a gene encoding 53BP1 using CRISPR-Cas gene editingcomprises contacting a cell with Cas9 polypeptide and a gRNA targetingthe 53BP1 gene locus. In some embodiments, gRNA sequence targeting the53BP1 gene locus is designed using the 53BP1 gene sequence using methodsknown in the art (see e.g., Briner (2014) Molecular Cell 56:333-339). Insome embodiments, gRNAs targeting the 53BP1 gene locus create indels inthe region of the 53BP1 gene that disrupt expression of 53BP1 in thecell. In some embodiments, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%,60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%,80-90%, or 90-100% of cells in the edited population lack detectableexpression of 53BP1.

In some embodiments, a 53BP1 inhibitor of the disclosure comprises asmall interfering RNA (siRNA) which silences 53BP1 expression. Methodsof silencing 53BP1 expression using siRNA are taught by US 2019/0010196which is incorporated by reference herein. Methods of delivering siRNAcan be performed using non-viral or viral delivery methods as describedin the art (e.g., Gao (2009) Mol Pharm 6:651-658; Oliveira (2006) JBiomed Biotechnol 2006:63675; Tatiparti (2017) Nanomaterials 7:77). Insome embodiments, a cell is transfected with siRNA targeting 53BP1mRNAs. In some embodiments, expression of 53BP1 is decreased by about50%, by about 60%, by about 70%, by about 80%, by about 90%, or by about100% following transfection with siRNA targeting 53BP1 mRNA.

Inhibition of DNA-PKcs

In some embodiments, the disclosure provides methods for increasing HDRof a DSB mediated by a site-directed nuclease in a target gene in a cellor population of cells, such as a quiescent cell that has been inducedto divide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by inhibition of DNA-PKcs. In someembodiments, the disclosure provides methods for increasing HDR of a DSBmediated by a site-directed nuclease in a cell or population of cellsexpressing an E6V mutation in HBB by inhibition of DNA-PKcs. In someembodiments, the disclosure provides methods for increasing HDR of a DSBmediated by a site-directed nuclease in a target gene in a cell orpopulation of cells, such as a quiescent cell that has been induced todivide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by inhibition of 53BP1 and DNA-PKcs. In someembodiments, the disclosure provides methods for increasing HDR of a DSBmediated by a site-directed nuclease in a cell or population of cellsexpressing an E6V mutation in HBB by inhibition of 53BP1 and DNA-PKcs.

The DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3)kinase-like kinase family (PIKK) and is a key kinase involved in NHEJrepair. DNA-PKcs is directed to DSB sites by binding to the Ku70/80heterodimer that has high-affinity for broken dsDNA ends and is firstrecruited to DSB sites. The complex formed at the DSB comprising DNA,Ku70/80 and DNA-PKcs is referred to as “DNA-PK” (Gottlieb (1993) Cell72:131-142). The large DNA-PK complex is responsible for holding the twoends of a broken DNA molecule together. Additionally, binding ofDNA-PKcs to the DNA-Ku70/80 complex results in activation of DNA-PKcskinase activity (Yoo et al (1999) Nucleic Acids Res 27:4679-4686; Calsou(1999) J Biol Chem 274:7848-7856). DNA-PKcs phosphorylates numerous NHEJrepair factors, thus enabling their function in NHEJ repair.

Accordingly, the present disclosure provides DNA-PKcs inhibitors thatinhibit NHEJ and promote HDR repair of a DSB in a target gene. In someembodiments, a DNA-PKcs inhibitor of the disclosure inhibits, reduces,disrupts, or blocks the ability of DNA-PKcs to a DSB site. In someembodiments, a DNA-PKcs inhibitor of the disclosure inhibits, reduces,disrupts, or blocks the ability of DNA-PKcs to bind to Ku70/80 to form aDNA-PK complex. In some embodiments, a DNA-PKcs inhibitor of thedisclosure inhibits, reduces, disrupts, or blocks the function of theDNA-PKcs kinase domain. In some embodiments, a DNA-PKcs inhibitor of thedisclosure inhibits, reduces, disrupts, or blocks phosphorylation ofNHEJ factors by the DNA-PKcs kinase domain. In some embodiments, aDNA-PKcs inhibitor of the disclosure is a polypeptide. In someembodiments, a DNA-PKcs inhibitor is a nucleic acid. In someembodiments, a DNA-PKcs inhibitor is a small molecule. In someembodiments, a DNA-PKcs inhibitor of the disclosure is a small moleculethat inhibits, disrupts or blocks the DNA-PKcs kinase domain.

In some embodiments, a DNA-PKcs inhibitor of the disclosure isidentified by binding affinity for DNA-PKcs or a fragment thereof (e.g.,a functional domain of DNA-PKs). Methods of measuring binding affinityof an inhibitor for a protein domain are known in the art. Non-limitingexamples include measuring inhibitor affinity by enzyme-linkedimmunosorbent assay (e.g., ELISA), immunoblot, immunoprecipitation-basedassay, fluorescence polarization assay, fluorescence resonance energytransfer assay, fluorescence anisotropy assay, yeast surface display(Gai (2007) Curr Opin Struct Biol 17:467-473), kinetic exclusion assay,surface plasmon resonance, or isothermal titration calorimetry.

In some embodiments, a DNA-PKcs inhibitor of the disclosure binds to theDNA-PKcs polypeptide. Methods of determining the structural interactionsthat enable binding of the inhibitor with the DNA-PKcs polypeptide areknown in the art. Non-limiting examples include X-ray crystallography,nuclear magnetic resonance (NMR) spectroscopy, electron microscopy,small-angle X-ray scattering (SAXS), and small-angle neutron scattering(SANS). In some embodiments, the structural interactions are determinedby a mutagenesis experiment wherein residues of the DNA-PKcs polypeptideare mutated and the effect on inhibitor binding are evaluated. Suchmethods enable identification of key residues that contribute tobinding.

In some embodiments, a method of inhibition of DNA-PKcs function in acell comprises contacting the cell with a small molecule inhibitor ofDNA-PKcs. In some embodiments, the DNA-PKCs inhibitor of the disclosureis a small molecule inhibitor Nu7441 (e.g., Leahy (2004) Bioorg Med ChemLett 14:6083-6087). In some embodiments, the DNA-PKcs inhibitor of thedisclosure is a PI 3-kinase inhibitor LY294002, which has been found toinhibit DNA-PKcs function in vitro (Izzard (1999) Cancer Res59:2581-2586). In some embodiments, the DNA-PKCs inhibitor of thedisclosure is a small molecule inhibitor capable of selectivelyinhibiting the activity of DNA-PKcs compared to PI 3-kinase.Non-limiting examples include 2-amino-chromen-4-ones that are describedby WO 03/024949, which is incorporated by reference herein. In someembodiments, the DNA-PKCs inhibitor of the disclosure is a smallmolecule inhibitor of DNA-PKcs function, including 1(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone (e.g., Kashishian (2003)Mol Cancer Ther 2:1257-1264). In some embodiments, the DNA-PKCsinhibitor of the disclosure is a small molecule inhibitor of DNA-PKcsfunction SU11752 (e.g., Ismail (2004) Oncogene 23:873-882). In someembodiments, the DNA-PKCs inhibitor of the disclosure is a smallmolecule inhibitor of DNA-PKcs function described in U.S. Pat. No.9,592,232, incorporated herein by reference. In some embodiments, theDNA-PKcs inhibitor of the disclosure is a small molecule inhibitor ofDNA-PKcs function described in U.S. Pat. No. 7,402,607, incorporatedherein by reference. In some embodiments, the DNA-PKCs inhibitor of thedisclosure is a small molecule inhibitor of DNA-PKcs function describedin U.S. Pat. No. 6,893,821, incorporated herein by reference. In someembodiments, the DNA-PKcs inhibitor of the disclosure is a smallmolecule inhibitor of DNA-PKcs function described in US 2018/0194782.

In some embodiments, the DNA-PKcs inhibitor of the disclosure isCompound 984 or Compound 296 described in U.S. Pat. No. 9,592,232. Thestructures of Compound 984 and Compound 296 are provided below:

Inhibition of Other Targets

In some embodiments, the disclosure provides methods for increasing HDRof a DSB mediated by a site-directed nuclease in a target gene in a cellor population of cells, such as a quiescent cell that has been inducedto divide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by inhibition of the NHEJ pathway, alone or incombination with inhibition of 53BP1 and/or DNA-PKcs. In someembodiments, the disclosure provides methods for increasing HDR of a DSBmediated by a site-directed nuclease in a target gene in a cell orpopulation of cells expressing an E6V mutation in the HBB gene, byinhibition of the NHEJ pathway, alone or in combination with inhibitionof 53BP1 and/or DNA-PKcs. In some embodiments, the disclosure provides amethod of inhibiting the NHEJ pathway by inhibition of key NHEJ enzymes.For example, in some embodiments, the disclosure provides a method ofinhibiting the NHEJ pathway by inhibition of Ku70/80. In someembodiments, the disclosure provides inhibitors of Ku70/80 includingCYREN (e.g., Arnoult (2017) Nature 549:548-552). In some embodiments,the disclosure provides a method of inhibiting the NHEJ pathway byinhibition of DNA Ligase IV. In some embodiments, the disclosureprovides inhibitors of DNA Ligase IV, including Scr7 (Maruyama (2015)Nat Biotechnol 33:538-542).

In some embodiments, the disclosure provides methods of increasing orimproving repair of a DNA DSB by HDR by inhibition of the MMEJ pathway(e.g., methods of MMEJ inhibition reviewed in Sfeir (2015) 40:701-714).In some embodiments, the disclosure provides methods of inhibition ofthe MMEJ pathway by inhibition of DNA polymerase theta (Pol θ). In someembodiments, the disclosure provides method of inhibition of the MMEJpathway by inhibition of PARP. In some embodiments, the disclosureprovides PARP inhibitors, including molecules developed for thetreatment of cancer, including Veliparib and Olaparib. In someembodiments, inhibition of the MMEJ pathway comprises inhibition ofMRE11. In some embodiments, the disclosure provides MRE11 inhibitors,including Mirin and derivatives (e.g., Shibata (2014) Molec Cell53:7-18).

In some embodiments, the disclosure provides methods for increasing HDRof a DSB mediated by a site-directed nuclease in a target gene in a cellor population of cells, such as a quiescent cell that has been inducedto divide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by treatment of a cell or population of cellswith a compound that stimulates HDR efficiency. In some embodiments, thedisclosure provides methods for increasing HDR of a DSB mediated by asite-directed nuclease in a target gene in a cell or populationexpressing an E6V mutation in the HBB gene, by treatment of a cell orpopulation of cells with a compound that stimulates HDR efficiency. Insome embodiments, the disclosure provides a stimulator of HDR, whereinthe stimulator of HDR is an agonist that promotes the function of afactor in the HDR pathway. In some embodiments, the disclosure providesa stimulator of an HDR factor, wherein the HDR factor is RAD51. In someembodiments, the disclosure provides agonists of RAD51, including RS-1(e.g., Jayathilaka (2008) PNAS 105:15848-15853).

Combination of Inhibitors

In some embodiments, the disclosure provides methods for increasing HDRof a DSB mediated by a site-directed nuclease in a target gene in a cellor population of cells, such as a quiescent cell that has been inducedto divide or a population of quiescent cells that has been induced todivide, e.g., CD34+ HSCs, by treatment with an inhibitor of 53BP1 incombination with an inhibitor of the NHEJ pathway. In some embodiments,the disclosure provides methods for increasing HDR of a DSB mediated bya site-directed nuclease in a target gene in a cell or population ofcells expressing an E6V mutation in the HBB gene, by treatment with aninhibitor of 53BP1 in combination with an inhibitor of the NHEJ pathway.In some embodiments, a method of increasing HDR is treatment with aninhibitor of 53BP1 in combination with an inhibitor of DNA-PKcs. In someembodiments, a method of increasing HDR is treatment with a polypeptideinhibitor of 53BP1 in combination with an inhibitor of DNA-PKcs. In someembodiments, a method of increasing HDR is treatment with a polypeptideinhibitor of 53BP1 comprising the amino acid sequence identified by SEQID NO: 70 in combination with a small molecule inhibitor of DNA-PKcs. Insome embodiments, a method of increasing HDR is treatment with apolypeptide inhibitor of 53BP1 comprising the amino acid sequenceidentified by SEQ ID NO: 70 in combination with Compound 984 or Compound296.

In some embodiments, a method of increasing HDR is treatment with aninhibitor of 53BP1 in combination with an inhibitor of Ku70/80. In someembodiments, a method of increasing HDR is treatment with a polypeptideinhibitor of 53BP1 comprising the amino acid sequence identified by SEQID NO: 70 in combination with an inhibitor of Ku70/80. In someembodiments, a method of increasing HDR is treatment with an inhibitorof 53BP1 in combination with an inhibitor of DNA Ligase IV. In someembodiments, a method of increasing HDR is treatment with a polypeptideinhibitor of 53BP1 comprising the amino acid sequence identified by SEQID NO: 70 in combination with an inhibitor of DNA Ligase IV.

In some embodiments, a method of increasing HDR is treatment with aninhibitor of 53BP1 in combination an inhibitor of the MMEJ pathway. Insome embodiments, a method of increasing HDR is treatment with apolypeptide inhibitor of 53BP1 comprising the amino acid sequenceidentified by SEQ ID NO: 70 in combination with an inhibitor of the MMEJpathway. In some embodiments, a method of increasing HDR is treatmentwith a polypeptide inhibitor of 53BP1 comprising the amino acid sequenceidentified by SEQ ID NO: 70 in combination with an inhibitor of PARP. Insome embodiments, a method of increasing HDR is treatment with apolypeptide inhibitor of 53BP1 comprising the amino acid sequenceidentified by SEQ ID NO: 70 in combination with an inhibitor of DNApolymerase theta.

Engineered Human Cells

Provided herein are methods of gene-editing within an HBB gene by repairof a DNA DSB in the HBB gene by the HDR pathway using a donorpolynucleotide. In some embodiments, the HBB gene is edited to correct amutation (e.g., an E6V mutation). In some embodiments, the HBB gene isedited by replacement with a different polynucleotide sequence, such asa polynucleotide sequence encoding a different gene (e.g., a transgene)or a variant version of the HBB gene. In some embodiments, the HBB geneis edited by deletion and insertion of a different gene (e.g., atransgene). In some embodiments, the HBB gene is edited by insertion ofa transgene comprising one or more exons and one or more introns. Insome embodiments, the HBB gene is edited by insertion of insertion of atransgene comprising only exons.

In some embodiments, an HBB gene is edited using methods herein tocorrect a genetic mutation that results in a monogenic disease. Amonogenic disease is characterized by a mutation in a single gene.Non-limiting examples of gene mutations that result in monogenic diseaseinclude mutation of the beta-globin (e.g., hemoglobin beta, HBB) genethat results in hemoglobinopathies. Non-limiting examples of disordersassociated with the HBB that are edited using methods described hereinare detailed in Table 1.

TABLE 1 Disorders Associated with Mutations in HBB Gene MonogenicDisorder Target Gene Sickle Cell Disease Hemoglobin subunit beta (HBB)Beta-Thalassemia Hemoglobin subunit beta (HBB)

In some embodiments, a monogenic disease is treated by administeringgene-edited human cells to a patient. In some embodiments, human cellsare taken from the patient and edited to correct a genetic mutationprior to being reintroduced to the patient for treatment of a monogenicdisorder. In some embodiments, cells from a patient are somatic cellsthat are reprogrammed to generated induced pluripotent stem cells(iPSCs). In some embodiments, iPSCs are gene-edited to correct amutation and then differentiated prior to administration to a patient.In some embodiments, cells from a patient are hematopoietic stem cells(HSCs) or hematopoietic progenitor cells (HPCs). In some embodiments,HSCs and HPCs are gene-edited and introduced to a patient for treatmentof a monogenic disease.

In some embodiments, HSCs are engineered (e.g., gene-edited) fortreatment of a hemoglobinopathy. Hemoglobinopathies encompass a numberof anemias that are associated with changes in the geneticallydetermined structure or expression of hemoglobin. These include changesto the molecule structure of the hemoglobin chain, such as occurs withsickle cell anemia, as well as changes in which synthesis of one or morechains is reduced or absent, such as occurs with various thalassemias.

Disorders specifically associated with the β-globin protein are referredto generally as β-hemoglobinopathies. For example, β-thalassemias resultfrom a partial or complete defect in the expression of the β-globingene, leading to deficient or absent hemoglobin A (HbA). HbA is the mostcommon human hemoglobin tetramer and consists of two α-chains and twoβ-chains (α₂β₂). β-thalassemias are due to mutations on the adultβ-globin gene (HBB) on chromosome 11, and are inherited in an autosomal,recessive fashion.

Sickle cell disease (SCD) includes SCA, sickle hemoglobin C disease,sickle beta-plus-thalassemia, and sickle beta-zero-thalassemia. Allforms of SCD are caused by mutations within the HBB gene. SCA is causedby a single missense mutation in the sixth codon (e.g., seventh codonwhen including the start codon) of the HBB gene (e.g., A to T),resulting in a substitution of glutamic acid by valine (e.g., Glu toVal). The mutant protein, when incorporated into hemoglobin, results inunstable hemoglobin HbS (α₂β₂ ^(S)) in contrast to normal adulthemoglobin HbA (α₂β₂ ^(A)). When HbS is the predominant form ofhemoglobin, it results in red blood cells (RBCs) with distorted sickleshape. Sickled RBCs are less flexible than normal RBCs, and tend to getstuck in small blood vessels, resulting in vaso-occlusive events. Theseevents are associated with tissue ischemia leading to acute and chronicpain.

In some embodiments, a patient is treated with gene-edited human cellsto ameliorate a hemoglobinopathy (e.g., de Montalembert (2008) BMJ,337:a1397; Sheth, et al. (2013) British J. Haematology 162:455-464).Methods towards treatment of hemoglobinopathies by production ofgenome-edited stem cells, including hematopoietic stem cells (HSCs), aretaught by US 2018/0030438 and US 2018/0200387 which are incorporated byreference herein. In some embodiments, a method of treating a patientwith hemoglobinopathy comprises administering gene-edited stem cells tothe patient that give rise to a population of circulating RBCs that willbe effective in ameliorating one or more clinical conditions associatedwith the patient's disease. In some embodiments, a gene-edited stem cellis an HSC, long-term repopulating hematopoietic cell or an LT-HSPC. Insome embodiments, a gene-edited HSC or HPC administered for treatment ofa hemoglobinopathy comprises a gene-edit within the HBB locus forcorrection of a mutation.

Engineered Hematopoietic Stem Cells

In some embodiments, stem cells are engineered (e.g., gene-edited) usingmethods of the disclosure. In some embodiments, stem cells areengineered to correct a gene mutation and/or replace a target gene. Insome embodiments, stem cells are engineered to correct an E6V mutationin an HBB gene. In some embodiments, engineered stem cells areadministered to a patient for treatment of a monogenic disease. In someembodiments, a stem cell comprises an HSC. In some embodiments, a stemcell comprises an HSC comprising an HBB gene encoding an E6V mutation.HSCs are defined by their pluripotency (e.g., capacity of a single HSCto generate any type of blood cell) and ability to self-renew. HSCs arecomprised of two populations: short-term HSCs and long-term HSCs. Shortterm HSCs are capable of self-renewal for a short period of time, whileLT-HSPCs are capable of indefinite self-renewal. LT-HSPCs are largely ina quiescent state, dividing only once every 145 days (Wilson, A. et al.(2008) Cell 135:1118-1129). In some embodiments, an HSC dividesasymmetrically wherein one daughter cell remains in a stem state and onedaughter cell expresses a distinct function or phenotype. In someembodiments, an HSC divides symmetrically wherein both daughter cellsretain a stem state.

Early descendants of an HSC are termed hematopoietic progenitor cells.Hematopoietic progenitor cells (HPCs) retain the ability todifferentiate into other cell types, but are not capable ofself-renewal. In some embodiments, progenitor cells of an HSC aredifferentiated cells. In some embodiments, progenitor cells of an HSCcomprise the same differentiation state. In some embodiments, progenitorcells of an HSC comprise different differentiation states. In someembodiments, progenitor cells of an HSC are lineage restricted precursorcells (e.g., a common myeloid progenitor cell, a common lymphoidprogenitor cell). In some embodiments, lineage restricted precursorcells further differentiate. In some embodiments, an HSC differentiatesinto a common lymphoid progenitor cell that further differentiates intocell types comprising B cells, natural killer (NK) cells, and T cells.In some embodiments, an HSC differentiates into a common myeloidprogenitor cell that further differentiates into cell types comprisingdendritic cells (DCs), monocytes, myeloblasts, monocyte-derived DCs,macrophages, neutrophils, eosinophils, basophils,megakaryocyte-erythroid progenitor cells, erythrocytes, megakaryocytes,and platelets.

In some embodiments, an HSC of the disclosure has positive expressionfor the cell surface marker CD34. In some embodiments, an HSC of thedisclosure has positive expression for cell surface markers comprisingCD38, CD45RA, CD90, c-Kit tyrosine kinase receptor, stem cell antigen-1(Sca-1), CD133 and CD49f. In some embodiments, an HSC of the disclosurehas negative or low expression for cell surface markers comprising CD38,CD45RA, CD90, Thy-1.1 cell surface antigen and CD49f. In someembodiments, an HSC of the disclosure has negative or low expression oflineage cell surface markers comprising CD2, CD3, CD11b, CD11c, CD14,CD16, CD19, CD24, CD56, CD66b, CD235. In some embodiments, an HSC of thedisclosure is an LT-HSC. In some embodiments, an LT-HSC has negative orlow expression of lineage cell surface markers comprising CD2, CD3,CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, CD235. In someembodiments, an LT-HSC has negative or low expression of cell surfacemarkers comprising CD45RA and CD38. In some embodiments, an LT-HSC haspositive expression for cell surface markers comprising CD34 and CD90.

Methods for isolation of HSCs are known in the art as taught by U.S.Pat. Nos. 5,643,741, 5,087,570, 5,677,136, 7,790,458, 10,006,004,10,086,045, 7,939,057, 10,058,573 that are incorporated by referenceherein. In some embodiments, a population of cells comprising HSCs isderived from the patient (e.g., an autologous HSC). In some embodiments,a population of cells comprising HSCs is derived from a healthy donor(e.g., an allogenic HSC). In some embodiments, a population of cellscomprising HSCs is derived from human cord blood. In some embodiments, apopulation of cells comprising HSCs is derived from bone marrow. In someembodiments, a population of cells comprising HSCs is derived from humanperipheral blood.

In some embodiments, a population of cells comprising HSCs is derivedfollowing treatment of a subject (e.g., a patient, a healthy donor) witha stem cell mobilizer. In some embodiments, a stem cell mobilizercomprises a CXCR4 antagonist. The chemokine stromal cell derivedfactor-1 (e.g., CXCL12) is a chemokine that binds to CXCR4 on HSCs andHPCs and signals for retention in the bone marrow. By blocking thisinteraction with a CXCR4 antagonist, HSCs and HPCs rapidly mobilize tothe blood (Broxmeyer, et al. (2005) J. Exp Med 18:1307-1318; Devine, S.et al (2008) Blood 112:990-998). Non-limiting examples of a CXCR4antagonist include TG-0054 (TaiGen Biotechnology, Co., Ltd. (Taipei,Taiwan)), AMD3465, AMD3100 (e.g., wherein AMD or AMD3100 is usedinterchangeably with plerixafor, rINN, USAN, JM3100, and its trade name,Mozobil™, see U.S. Pat. Nos. 6,835,731 and 6,825,351), and NIBR1816(Novartis, Basil, Switzerland). In some embodiments, a stem-cellmobilizer is plerixafor.

In some embodiments, a stem cell mobilizer comprises a colonystimulating factor. Non-limiting examples of a colony stimulating factorinclude, but are not limited to, granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), stem cell factor (SCF),FLT-3 ligand, or a combination thereof. Use of G-CSF as a stem cellmobilizing factor has demonstrated increased yield of stem cells fromperipheral blood (Morton, et al (2001) Blood 98:3186; Smith, T. et al.(1997) J. Clin. Oncol. 15:5-10) In some embodiments, a stem cellmobilizer is a combination of a CXCR4 antagonist and a colonystimulating factor. In some embodiments, a stem cell mobilizer is acombination of Plerixafor and G-CSF.

In some embodiments, CD34+ HSCs are enriched following isolation from asubject (e.g., a patient, a healthy donor). In some embodiments, CD34+HSCs are enriched from human blood, bone marrow, or cord blood. Methodsof enriching CD34+ HSCs are known in the art. In some embodiments, CD34+HSCs are enriched using a magnetic cell separator. In some embodiments,CD34+ HSCs are enriched by fluorescent activated cell sorting (FACS). Insome embodiments, CD34+ HSCs are enriched by magnetic bead sorting forcells expressing CD34.

In some embodiments, an enriched population of CD34+ cells has a purityof at least 40%, at least 50%, at least 60%, at least 70%, at least 80%,at least 90%, or at least 100%. In some embodiments, an enrichedpopulation of CD34+ cells has a purity of at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100%. In someembodiments, an enriched population of CD34+ cells has a purity of atleast about 90%. In some embodiments, an enriched population of CD34+cells has a purity of at least about 91%. In some embodiments, anenriched population of CD34+ cells has a purity of at least about 92%.In some embodiments, an enriched population of CD34+ cells has a purityof at least about 93%. In some embodiments, an enriched population ofCD34+ cells has a purity of at least about 94%. In some embodiments, anenriched population of CD34+ cells has a purity of at least about 95%.In some embodiments, an enriched population of CD34+ cells has a purityof at least about 96%. In some embodiments, an enriched population ofCD34+ cells has a purity of at least about 97%. In some embodiments, anenriched population of CD34+ cells has a purity of at least about 98%.In some embodiments, an enriched population of CD34+ cells has a purityof at least about 99%. In some embodiments, an enriched population ofCD34+ cells has a purity of at least about 100%.

In some embodiments, an enriched population of CD34+ cells comprisesLT-HSPCs. In some embodiments, the proportion of the CD34+ populationthat is LT-HSPCs is 0.01-0.05%, 0.01-0.1%, 0.05-0.1%, 0.05-1%, 0.1-0.5%,0.1-0.7%, 0.1-1.0%, 0.1-1.5%, 0.1-2.0%, 0.5-1.5%, 0.5-2.0%, or 1-2%. Insome embodiments, the proportion of the CD34+ population that isLT-HSPCs is 0.05-1%. In some embodiments, the proportion of the CD34+population that is LT-HSPCs is 0.1-1%. In some embodiments, theproportion of the CD34+ population that is LT-HSPCs is 0.1-2%. In someembodiments, the proportion of the CD34+ population that is LT-HSPCs isat least about 0.01%, at least about 0.05%, at least about 0.1%, atleast about 0.2%, at least about 0.3%, at least about 0.4%, at leastabout 0.5%, at least about 0.6%, at least about 0.7%, at least about0.8%, at least about 0.9%, or at least about 1.0% of the population.

In some embodiments, gene-editing of HSCs is performed prior toenrichment of CD34+HSCs. In some embodiments, gene-editing of HSCs isperformed following enrichment of CD34+ HSCs. In some embodiments,following gene-editing, a method is used to selected for gene-editedHSCs from a population comprising CD34+ HSCs. In some embodiments, amethod of isolating gene-edited HSCs enrichment of HSCs expressingtruncated nerve growth factor (tNGFR) as described in the art (Dever etal (2016) Nature 539:384-389).

For ex vivo therapy, transplantation requires clearance of bone-marrowniches for donor HSCs to engraft. Methods are known in the art fordepletion of the bone-marrow niche, including methods of treating withradiation, chemotherapy or a combination thereof.

Engineered Induced Pluripotent Stem Cells

In some embodiments, genetically engineered human cells of thedisclosure are derived from induced pluripotent stem cells (iPSCs).iPSCs are reprogrammed from somatic cells to a pluripotent state whereinthey can differentiate into all three germ layers. An advantage of usingiPSCs is that the cell can be derived from the same subject to which theprogenitor cells are to be administered. That is, a somatic cell can beobtained from a subject, reprogrammed to an iPSC, and thenre-differentiated into a progenitor cell to be administered to thesubject for treatment of a disorder (e.g., an autologous progenitor).Since the progenitors are derived from an autologous source, the risk ofengraftment rejection or allergic responses is reduced compared to theuse of cells form another subject or group of subjects. Thus, an iPSCcan be gene-edited and reintroduced into a patient for correction of adisease resulting from a somatic genetic mutation.

Briefly, human iPSCs can be obtained by transducing somatic cells withstem cell associated transcription factors that include OCT4, SOX2, andNANOG (Budniatzky et al. (2014) Stem Cells Transl Med 3:448-457; Barretet al. Stem Cells Trans Med (2014) 3:1-6; Focosi et al. (2014) BloodCancer Journal 4:e211). Exemplary methods for reprogramming somaticcells to generate iPSCs are known in the art as described by US2019/0038771 which is incorporated by reference herein.

Pharmaceutical Compositions

The present disclosure includes pharmaceutical compositions comprising adonor polynucleotide, a gRNA, and a Cas9 protein, in combination withone or more pharmaceutically acceptable excipient, carrier or diluent.In some embodiments, the disclosure provides pharmaceutical compositionscomprising a donor polynucleotide or recombinant vector, a gRNA, a Cas9protein, and a 53BP1 inhibitor and/or DNA-PKcs inhibitor, in combinationwith one or more pharmaceutically acceptable excipient, carrier ordiluent. In particular embodiments, the donor polynucleotide isencapsulated in a nanoparticle, e.g., a lipid nanoparticle. In someembodiments, the gRNA is encapsulated in a nanoparticle. In someembodiments, a Cas nuclease (e.g., SpCas9) is encapsulated in ananoparticle. In some embodiments, the 53BP1 inhibitor is encapsulatedin a nanoparticle, e.g., a lipid nanoparticle. In some embodiments, theDNA-PKcs inhibitor is encapsulated in a nanoparticle, e.g., a lipidnanoparticle. In some embodiments, the donor polynucleotide, gRNA, Cas9protein, 53BP1 inhibitor and/or DNAK-PKcs inhibitor are encapsulated inthe same or different nanoparticle, e.g., lipid nanoparticle. Inparticular embodiments, an mRNA encoding a Cas nuclease or nanoparticleencapsulating a Cas nuclease is present in a pharmaceutical composition.In various embodiments, the one or more mRNA present in thepharmaceutical composition is encapsulated in a nanoparticle, e.g., alipid nanoparticle. In particular embodiments, the molar ratio of thefirst mRNA to the second mRNA is about 1:50, about 1:25, about 1:10,about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1.

In some embodiments, the ratio between the lipid composition and thedonor polynucleotide can be about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1,24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1,36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1,48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the lipidcomposition to the polynucleotide is about 20:1 or about 15:1.

In one embodiment, the lipid nanoparticles described herein can comprisepolynucleotides (e.g., donor polynucleotide) in a lipid:polynucleotideweight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1,50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, butnot limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, fromabout 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 toabout 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1,from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, fromabout 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, fromabout 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1to about 20:1, from about 15:1 to about 25:1, from about 15:1 to about30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, fromabout 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about70:1.

In one embodiment, the lipid nanoparticles described herein can comprisethe polynucleotide in a concentration from approximately 0.1 mg/ml to 2mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml,1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

Methods of Treatment

Provided herein are methods of treating a patient with a disease bygene-editing a genomic DNA molecule, such as correcting a mutation in agenomic DNA molecule. In some embodiments, the method may compriseintroducing a donor polynucleotide, system, vector, or pharmaceuticalcomposition described herein into a cell. In some embodiments, themethod may comprise administering a donor polynucleotide or recombinantvector, system, vector, or pharmaceutical composition to a subject inneed thereof (e.g., a patient having a disease caused by a mutation).

In some embodiments, the disclosure provides methods of treating apatient with a disease associated with a mutation in the HBB gene. Insome embodiments, the mutation in the HBB gene is E6V. In someembodiments, the disease associated with a mutation in the HBB gene is asickle cell disease (SCD, also referred to as sickle cell anemia orSCA). In some embodiments, the disease associated with a mutation in theHBB gene is a β-thalassemia.

Embodiments of the disclosure encompass methods for editing a targetnucleic acid molecule (a genomic DNA) in a cell. In some embodiments,the method comprises introducing a donor polynucleotide described hereininto a cell. In some embodiments, the method comprises contacting thecell with a pharmaceutical composition described herein. In someembodiments, the method comprises generating a stable cell linecomprising a targeted edited nucleic acid molecule. In some embodiments,the cell is a eukaryotic cell. Non-limiting examples of eukaryotic cellsinclude yeast cells, plant cells, insect cells, cells from aninvertebrate animal, cells from a vertebrate animal, mammalian cells,rodent cells, mouse cells, rat cells, and human cells. In someembodiments, the eukaryotic cell may be a mammalian cell. In someembodiments, the eukaryotic cell may be a rodent cell. In someembodiments, the eukaryotic cell may be a human cell. Similarly, thetarget sequence may be from any such cells or in any such cells.

The donor polynucleotide, system, vector, or pharmaceutical compositiondescribed herein may be introduced into the cell via any methods knownin the art, such as, e.g., viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran-mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, shear-driven cell permeation, fusion to acell-penetrating peptide followed by cell contact, microinjection, andnanoparticle-mediated delivery. In some embodiments, the vector systemmay be introduced into the cell via viral infection. In someembodiments, the vector system may be introduced into the cell viabacteriophage infection.

Embodiments of the invention also encompass treating a patient withdonor polynucleotide or recombinant vector, system, vector, orpharmaceutical composition described herein. In some embodiments, thepatient has a mutation in the HBB gene. In some embodiments, the patienthas an E6V mutation in the HBB gene. In some embodiments, the method maycomprise administering the donor polynucleotide, system, vector, orpharmaceutical composition described herein to the patient. The methodmay be used as a single therapy or in combination with other therapiesavailable in the art. In some embodiments, the patient may have amutation (such as, e.g., insertion, deletion, substitution, chromosometranslocation) in a disease-associated gene. In some embodiments,administration of the donor polynucleotide, system, vector, orpharmaceutical composition may result in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of thedisease-associated gene in the patient. Certain embodiments may includemethods of repairing the patient's mutation in the disease-associatedgene. In some embodiments, the mutation may result in one or more aminoacid changes in a protein expressed from the disease-associated gene. Insome embodiments, the mutation may result in one or more nucleotidechanges in an RNA expressed from the disease-associated gene. In someembodiments, the mutation may alter the expression level of thedisease-associated gene. In some embodiments, the mutation may result inincreased or decreased expression of the gene. In some embodiments, themutation may result in gene knockdown in the patient. In someembodiments, the administration of the donor polynucleotide, system,vector, or pharmaceutical composition may result in the correction ofthe patient's mutation in the disease-associated gene. In someembodiments, the administration of the donor polynucleotide, system,vector, or pharmaceutical composition may result in gene knockout in thepatient. In some embodiments, the administration of the donorpolynucleotide, system, vector, or pharmaceutical composition system mayresult in replacement of an exon sequence, an intron sequence, atranscriptional control sequence, a translational control sequence, or anon-coding sequence of the disease-associated gene.

In some embodiments, the administration of the donor polynucleotide,system, vector, or pharmaceutical composition may result in integrationof an exogenous sequence (e.g., the donor polynucleotide sequence) intothe patient's genomic DNA. In some embodiments, the administration ofthe donor polynucleotide, system, vector, or pharmaceutical compositionresults in integration of an exogenous sequence encoding wild-type HBB(e.g., lacking the E6V mutation) into the patient's genomic DNA. In someembodiments, the administration of the donor polynucleotide, system,vector or pharmaceutical composition results in exchanging a region ofthe HBB gene correcting an E6V mutation for a region encoding the E6Vmutation. In some embodiments, the exogenous sequence may comprise aprotein or RNA coding sequence operably linked to an exogenous promotersequence such that, upon integration of the exogenous sequence into thepatient's genomic DNA, the patient is capable of expressing the proteinor RNA encoded by the integrated sequence. The exogenous sequence mayprovide a supplemental or replacement protein coding or non-codingsequence. For example, the administration of the donor polynucleotide,system, vector, or pharmaceutical composition may result in thereplacement of the mutant portion of the disease-associated gene in thepatient. In some embodiments, the mutant portion may include an exon ofthe disease-associated gene. In other embodiments, the integration ofthe exogenous sequence may result in the expression of the integratedsequence from an endogenous promoter sequence present on the patient'sgenomic DNA. For example, the administration of the donorpolynucleotide, system, vector, or pharmaceutical composition may resultin supply of a functional gene product of the disease-associated gene torectify the patient's mutation. In yet other embodiments, theadministration of the donor polynucleotide, system, vector, orpharmaceutical composition may result in integration of an exonsequence, an intron sequence, a transcriptional control sequence, atranslational control sequence, or a non-coding sequence into thepatient's genomic DNA.

Additional embodiments of the invention also encompass methods oftreating the patient in a tissue-specific manner. In some embodiments,the method may comprise administering the donor polynucleotide, system,vector, or pharmaceutical composition comprising a tissue-specificpromoter as described herein to the patient. Non-limiting examples ofsuitable tissues for treatment by the methods include the immune system,neuron, muscle, pancreas, blood, kidney, bone, lung, skin, liver, andbreast tissues.

In some embodiments, the disclosure provides a method to correct amutation in a genomic DNA molecule (gDNA) in a cell, the methodcomprising contacting the cell with a donor polynucleotide describedherein, a system comprising a donor polynucleotide, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein when the donor polynucleotide,system or composition contacts the cell, an HDR DNA repair pathwayinserts the donor polynucleotide into a double-stranded DNA breakintroduced into the gDNA at a location proximal to the mutation, therebycorrecting the mutation.

In some embodiments, the disclosure provides a method to correct amutation in a genomic DNA molecule (gDNA) in a cell, the methodcomprising contacting the cell with a donor polynucleotide orrecombinant vector described herein, a system comprising a donorpolynucleotide or recombinant vector, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, wherein when the donor polynucleotide, recombinantvector, system or composition contacts the cell, an HDR DNA repairpathway exchanges the donor polynucleotide or recombinant vector for acorresponding nucleic acid region in the HBB gene at a location proximalto the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method to correct amutation in a genomic DNA molecule (gDNA) in a cell, the methodcomprising contacting the cell with a donor polynucleotide orrecombinant vector described herein, a system comprising a donorpolynucleotide or recombinant vector, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, wherein when the donor polynucleotide, recombinantvector, system or composition contacts the cell, an HDR DNA repairpathway exchanges a region around a double-stranded DNA break introducedinto the gDNA at a location proximal to the mutation, thereby correctingthe mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising isolating a cell fromthe patient, contacting the cell with a donor polynucleotide describedherein, a system comprising a donor polynucleotide, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein, when the donor polynucleotide,system or composition contacts the cell, an HDR DNA repair pathwayinserts the donor polynucleotide into a double-stranded DNA breakintroduced into the gDNA at a location proximal to the mutation, therebycorrecting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising isolating a cell fromthe patient, contacting the cell with a donor polynucleotide orrecombinant vector described herein, a system comprising a donorpolynucleotide or recombinant vector, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, an HDR DNA repair pathway exchanges the donorpolynucleotide or recombinant vector for a corresponding nucleic acidregion in the HBB gene at a location proximal to the mutation, therebycorrecting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising isolating a cell fromthe patient, contacting the cell with a donor polynucleotide orrecombinant vector described herein, a system comprising a donorpolynucleotide or recombinant vector, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, an HDR DNA repair pathway exchanges a region around adouble-stranded DNA break introduced into the gDNA at a locationproximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising administering to thepatient an effective amount of a donor polynucleotide described herein,a system comprising a donor polynucleotide, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, wherein, when the donor polynucleotide, system orcomposition is administered, an HDR DNA repair pathway inserts the donorpolynucleotide into a double-stranded DNA break introduced into the gDNAat a location proximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising administering to thepatient an effective amount of a donor polynucleotide or recombinantvector described herein, a system comprising a donor polynucleotide orrecombinant vector, a gRNA, and a site-directed nuclease, according tothe disclosure, or a pharmaceutical composition described herein, an HDRDNA repair pathway exchanges the donor or recombinant vector acorresponding nucleic acid region in the HBB gene at a location proximalto the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease by correcting a mutation in a genomic DNAmolecule (gDNA) in a cell, the method comprising administering to thepatient an effective amount of a donor polynucleotide or recombinantvector, a gRNA, and a site-directed nuclease, according to thedisclosure, or a pharmaceutical composition described herein, an HDR DNArepair pathway exchanges a region around a double-stranded DNA breakintroduced into the gDNA at a location proximal to the mutation, therebycorrecting the mutation.

In some embodiments, the disclosure provides a method to correct an E6Vmutation in HBB in a cell comprising an HBB gene encoding the E6Vmutation, the method comprising contacting the cell with a donorpolynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein when the donor polynucleotide orrecombinant vector, system or composition contacts the cell, an HDR DNArepair pathway inserts the donor polynucleotide or recombinant vectorinto a double-stranded DNA break introduced into the gDNA at a locationproximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method to correct an E6Vmutation in HBB in a cell comprising an HBB gene encoding the E6Vmutation, the method comprising contacting the cell with a donorpolynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein when the donor polynucleotide,recombinant vector, system or composition contacts the cell, an HDR DNArepair pathway exchanges the donor polynucleotide or recombinant vectorfor a corresponding nucleic acid region in the HBB gene at a locationproximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method to correct an E6Vmutation in HBB in a cell comprising an HBB gene encoding the E6Vmutation, the method comprising contacting the cell with a donorpolynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein when the donor polynucleotide,recombinant vector, system or composition contacts the cell, an HDR DNArepair pathway exchanges a region around a double-stranded DNA breakintroduced into the gDNA at a location proximal to the mutation, therebycorrecting the mutation.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising isolating a cell from the patient, contacting the cell with adonor polynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein, when the donor polynucleotide,recombinant vector, system or composition contacts the cell, an HDR DNArepair pathway inserts the donor polynucleotide or recombinant vectorinto a double-stranded DNA break introduced into the gDNA at a locationproximal to the mutation, thereby correcting the E6V mutation andtreating the patient.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising isolating a cell from the patient, contacting the cell with adonor polynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, an HDR DNA repair pathway exchanges thedonor polynucleotide or recombinant vector for a corresponding nucleicacid region in the HBB gene at a location proximal to the mutation,thereby correcting the E6V mutation and treating the patient.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising isolating a cell from the patient, contacting the cell with adonor polynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, an HDR DNA repair pathway exchanges aregion around a double-stranded DNA break introduced into the gDNA at alocation proximal to the mutation, thereby correcting the E6V mutationand treating the patient.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising administering to the patient an effective amount of a donorpolynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, wherein, when the donor polynucleotide,recombinant vector, system or composition is administered, an HDR DNArepair pathway inserts the donor polynucleotide or recombinant vectorinto a double-stranded DNA break introduced into the gDNA at a locationproximal to the mutation, thereby correcting the E6V mutation andtreating the patient.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising administering to the patient an effective amount of a donorpolynucleotide or recombinant vector described herein, a systemcomprising a donor polynucleotide or recombinant vector, a gRNA, and asite-directed nuclease, according to the disclosure, or a pharmaceuticalcomposition described herein, an HDR DNA repair pathway exchanges thedonor polynucleotide or recombinant vector for a corresponding nucleicacid region in the HBB gene at a location proximal to the mutation,thereby correcting the E6V mutation and treating the patient.

In some embodiments, the disclosure provides a method of treating apatient with a disease associated with an E6V mutation in HBB bycorrecting the E6V mutation in the HBB gene in a cell, the methodcomprising administering to the patient an effective amount of a donorpolynucleotide or recombinant vector, a gRNA, and a site-directednuclease, according to the disclosure, or a pharmaceutical compositiondescribed herein, an HDR DNA repair pathway exchanges a region around adouble-stranded DNA break introduced into the gDNA at a locationproximal to the mutation, thereby correcting the E6V mutation andtreating the patient.

In some embodiments, the cell is a hematopoietic stem cell. In someembodiments, the cell is a hematopoietic stem cell comprising an HBBgene encoding an E6V mutation. In some embodiments, the cell is apatient-specific induced pluripotent stem cell (iPSC). In someembodiments, the cell is a patient-specific induced pluripotent stemcell (iPSC) comprising an HBB gene encoding an E6V mutation. In someembodiments, the method further comprises differentiating the iPSCcomprising the corrected mutation into a differentiated cell; andimplanting the differentiated cell into a patient. In some embodiments,treatment results in the translation of an mRNA transcribed from thegenomic DNA molecule (gDNA) comprising the inserted donorpolynucleotide, wherein the translation results in the formation of atranslation product (protein) that alleviates the disease or that doesnot cause or contribute to the disease.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant application shall control.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

About: As used herein, the term “about” (alternatively “approximately”)will be understood by persons of ordinary skill and will vary to someextent depending on the context in which it is used. If there are usesof the term which are not clear to persons of ordinary skill given thecontext in which it is used, “about” will mean up to plus or minus 10%of the particular value.

Amino acid: As used herein, the term “amino acid” refers to naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

Amino acid substitution: As used herein, an “amino acid substitution”refers to the replacement of at least one existing amino acid residue ina predetermined amino acid sequence (an amino acid sequence of astarting polypeptide) with a second, different “replacement” amino acidresidue. An “amino acid insertion” refers to the incorporation of atleast one additional amino acid into a predetermined amino acidsequence. While the insertion will usually consist of the insertion ofone or two amino acid residues, larger “peptide insertions,” can also bemade, e.g., insertion of about three to about five or even up to aboutten, fifteen, or twenty amino acid residues. The inserted residue(s) maybe naturally occurring or non-naturally occurring as disclosed above. An“amino acid deletion” refers to the removal of at least one amino acidresidue from a predetermined amino acid sequence.

Base Composition: As used herein, the term “base composition” refers tothe proportion of the total bases of a nucleic acid consisting ofguanine+cytosine or thymine (or uracil)+adenine nucleobases.

Base Pair: As used herein, the term “base pair” refers to twonucleobases on opposite complementary polynucleotide strands, or regionsof the same strand, that interact via the formation of specific hydrogenbonds. As used herein, the term “Watson-Crick base pairing”, usedinterchangeably with “complementary base pairing”, refers to a set ofbase pairing rules, wherein a purine always binds with a pyrimidine suchthat the nucleobase adenine (A) forms a complementary base pair withthymine (T) and guanine (G) forms a complementary base pair withcytosine (C) in DNA molecules. In RNA molecules, thymine is replaced byuracil (U), which, similar to thymine (T), forms a complementary basepair with adenine (A). The complementary base pairs are bound togetherby hydrogen bonds and the number of hydrogen bonds differs between basepairs. As in known in the art, guanine (G)-cytosine (C) base pairs arebound by three (3) hydrogen bonds and adenine (A)-thymine (T) or uracil(U) base pairs are bound by two (2) hydrogen bonds.

Base pairing interactions that do not follow these rules can occur innatural, non-natural, and synthetic nucleic acids and are referred toherein as “non-Watson-Crick base pairing” or alternatively“non-canonical base pairing”. A “wobble base pair” is a pairing betweentwo nucleobases in RNA molecules that does not follow Watson-Crick basepair rules. For example, inosine is a nucleoside that is structurallysimilar to guanosine, but is missing the 2-amino group. Inosine is ableto form two hydrogen bonds with each of the four natural nucleobases(Oda et al., (1991) Nucleic Acids Res 19:5263-5267) and it is often usedby researchers as a “universal” base, meaning that it can base pair withall the naturally-occurring or canonical bases. The four main wobblebase pairs are the guanine-uracil (G-U) base pair, thehypoxanthine-uracil (I-U) base pair, the hypoxanthine-adenine (I-A) basepair, and the hypoxanthine-cytosine (I-C) base pair. In order tomaintain consistency of nucleic acid nomenclature, “I” is used forhypoxanthine because hypoxanthine is the nucleobase of inosine;nomenclature otherwise follows the names of nucleobases and theircorresponding nucleosides (e.g., “G” for both guanine and guanosine—aswell as for deoxyguanosine). The thermodynamic stability of a wobblebase pair is comparable to that of a Watson-Crick base pair. Wobble basepairs play a role in the formation of secondary structure in RNAmolecules.

Blunt-end: As used herein, the term “blunt-end” “blunt-ended” refers tothe structure of an end of a duplexed or double-stranded nucleic acid(e.g., DNA), wherein both complementary strands comprising the duplexterminate, at least at one end, in a base pair. Hence, neither strandcomprising the duplex extends further from the end than the other.

Codon: As used herein, the term “codon” refers to a sequence of threenucleotides that together form a unit of genetic code in a DNA or RNAmolecule. A codon is operationally defined by the initial nucleotidefrom which translation starts and sets the frame for a run of successivenucleotide triplets, which is known as an “open reading frame” (ORF).For example, the string GGGAAACCC, if read from the first position,contains the codons GGG, AAA, and CCC; if read from the second position,it contains the codons GGA and AAC; and if read from the third position,GAA and ACC. Thus, every nucleic sequence read in its 5′→3′ directioncomprises three reading frames, each producing a possibly distinct aminoacid sequence (in the given example, Gly-Lys-Pro, Gly-Asn, or Glu-Thr,respectively). DNA is double-stranded defining six possible readingframes, three in the forward orientation on one strand and three reverseon the opposite strand. Open reading frames encoding polypeptides aretypically defined by a start codon, usually the first AUG codon in thesequence.

Corrects or induces a mutation: As used herein, the term “corrects orinduces a mutation” refers to a function of a donor polynucleotide, suchas those described herein, to incorporate a desired alteration into anucleotide sequence comprising a genomic DNA (gDNA) molecule uponinsertion of the donor polynucleotide into a double-strand break (DSB)induced in the gDNA molecule, thereby changing the nucleotide sequenceof the gDNA.

The term “corrects a mutation” refers to an incorporation of a desiredalteration by a donor polynucleotide that results in a change of one ormore nucleotides in a gDNA that comprises a mutation (e.g., adeleterious or disease-causing mutation) such that the mutation isreverted or transmuted in a desired manner. The identification of amutation to correct can be determined by comparison of the nucleotidesequence of a gDNA known, or suspected to, comprise the mutation to thenucleotide sequence of a wild-type gDNA.

The term “induces a mutation” refers to an incorporation of a desiredalteration by a donor polynucleotide that results in a change of one ormore nucleotides in a gDNA such that the gDNA is mutated in a desiredmanner. A mutation induced by a donor polynucleotide may be any type ofmutation known in the art. In some embodiments, the induction of amutation is for therapeutic purposes or results in a therapeutic effect.

Covalently linked: As used herein, the term “covalently linked”(alternatively “conjugated”, “linked,” “attached,” “fused”, or“tethered”), when used with respect to two or more moieties, means thatthe moieties are physically associated or connected with one another, bywhatever means including chemical conjugation, recombinant techniques orenzymatic activity, either directly or via one or more additionalmoieties that serves as a linking agent, to form a structure that issufficiently stable so that the moieties remain physically associatedunder the conditions in which the structure is used, e.g., physiologicalconditions.

Complementary: As used herein, the term “complementary” or“complementarity” refers to a relationship between the sequence ofnucleotides comprising two polynucleotide strands, or regions of thesame polynucleotide strand, and the formation of a duplex comprising thestrands or regions, wherein the extent of consecutive base pairingbetween the two strands or regions is sufficient for the generation of aduplex structure. It is known that adenine (A) forms specific hydrogenbonds, or “base pairs”, with thymine (T) or uracil (U). Similarly, it isknown that a cytosine (C) base pairs with guanine (G). It is also knownthat non-canonical nucleobases (e.g., inosine) can hydrogen bond withnatural bases. A sequence of nucleotides comprising a first strand of apolynucleotide, or a region, portion or fragment thereof, is said to be“sufficiently complementary” to a sequence of nucleotides comprising asecond strand of the same or a different nucleic acid, or a region,portion, or fragment thereof, if, when the first and second strands arearranged in an antiparallel fashion, the extent of base pairing betweenthe two strands maintains the duplex structure under the conditions inwhich the duplex structure is used (e.g., physiological conditions in acell). It should be understood that complementary strands or regions ofpolynucleotides can include some base pairs that are non-complementary.Complementarity may be “partial,” in which only some of the nucleobasescomprising the polynucleotide are matched according to base pairingrules. Or, there may be “complete” or “total” complementarity betweenthe nucleic acids. Although the degree of complementarity betweenpolynucleotide strands or regions has significant effects on theefficiency and strength of hybridization between the strands or regions,it is not required for two complementary polynucleotides to base pair atevery nucleotide position. In some embodiments, a first polynucleotideis 100% or “fully” complementary to a second polynucleotide and thusforms a base pair at every nucleotide position. In some embodiments, afirst polynucleotide is not 100% complementary (e.g., is 90%, or 80% or70% complementary) and contains mismatched nucleotides at one or morenucleotide positions. While perfect complementarity is often desired,some embodiments can include one or more but preferably 6, 5, 4, 3, 2,or 1 mismatches.

Contacting: As used herein, the term “contacting” means establishing aphysical connection between two or more entities. For example,contacting a cell with an agent (e.g., an RNA, a lipid nanoparticlecomposition, or other pharmaceutical composition of the disclosure)means that the cell and the agent are made to share a physicalconnection. Methods of contacting cells with external entities both invivo, in vitro, and ex vivo are well known in the biological arts. Inexemplary embodiments of the disclosure, the step of contacting amammalian cell with a composition (e.g., an isolated RNA, nanoparticle,or pharmaceutical composition of the disclosure) is performed in vivo.For example, contacting a lipid nanoparticle composition and a cell (forexample, a mammalian cell) which may be disposed within an organism(e.g., a mammal) may be performed by any suitable administration route(e.g., parenteral administration to the organism, including intravenous,intramuscular, intradermal, and subcutaneous administration). For a cellpresent in vitro, a composition (e.g., a lipid nanoparticle or anisolated RNA) and a cell may be contacted, for example, by adding thecomposition to the culture medium of the cell and may involve or resultin transfection. Moreover, more than one cell may be contacted by anagent.

Culture: As used herein, the term “culture” can be used interchangeablywith the terms “culturing”, “grow”, “growing”, “maintain”,“maintaining”, “expand”, “expanding” when referring to a cell culture orthe process of culturing. The term refers to a cell (e.g., a primarycell) that is maintained outside its normal environment (e.g., a tissuein a living organism) under controlled conditions. Cultured cells aretreated in a manner that enables survival. Culturing conditions can bemodified to alter cell growth, homeostasis, differentiation, division,or a combination thereof in a controlled and reproducible manner. Theterm does not imply that all cells in the culture survive, grow, ordivide as some may die, enter a state of quiescence, or enter a state ofsenescence. Cells are typically cultured in media, which can be changedduring the course of the culture. Components can be added to the mediaor environmental factors (e.g., temperature, humidity, atmospheric gaslevels) to promote cell survival, growth, homeostasis, division, or acombination thereof.

Denaturation: As used herein, the term “denaturation” refers to theprocess by which the hydrogen bonding between base paired nucleotides ina nucleic acid is disrupted, resulting in the loss of secondary and/ortertiary nucleic acid structure (e.g., the separation of previouslyannealed strands). Denaturation can occur by the application of anexternal substance, energy, or biochemical process to a nucleic acid.

Double-strand break: As used herein the term, “double-strand break”(DSB) refers to a DNA lesion generated when the two complementarystrands of a DNA molecule are broken or cleaved, resulting in two freeDNA ends or termini. DSBs may occur via exposure to environmentalinsults (e.g., irradiation, chemical agents, or UV light) or generateddeliberately (e.g., via a site-directed nuclease) and for a definedbiological purpose (e.g., the insertion of a donor polynucleotide tocorrect a mutation).

Duplex: As used herein, the term “duplex” refers to a structure formedby complementary strands of a double-stranded polynucleotide, orcomplementary regions of a single-stranded polynucleotide that foldsback on itself. The duplex structure of a nucleic acid arises as aconsequence of complementary nucleotide sequences being bound together,or hybridizing, by base pairing interactions.

EC₅₀: As used herein, the term “EC₅₀” refers to the concentration of acomposition which induces a response, either in an in vitro or an invivo assay, which is 50% of the maximal response, i.e., halfway betweenthe maximal response and the baseline.

Effective dose: As used herein, the term “effective dose” or “effectivedosage” is defined as an amount sufficient to achieve or at leastpartially achieve the desired effect.

Engraftment: As used herein, the term “engraftment” is usedinterchangeably with the term “chimerism” and refers to the processwherein donor stem cells are administered to (e.g., transplanted) ahost, traffic to a tissue compartment, and establish within thatcompartment by undergoing self-renewal and generating differentiatedcells for reconstitution of the tissue compartment. Often the termengraftment refers to the success of a hematopoietic stem cell (HSC)transplant (e.g., a bone marrow transplant). The term “engraftment” inthis context refers to the persistence of transplanted HSCs and theirprogenitors following administration. The term engraftment can alsorefer to the success of a T cell therapy wherein ex vivo manipulated Tcells are administered to a host. The term “engraftment” in this contextrefers to the persistence of transplanted donor T cells and theirprogenitors following administration

Genome editing: As used herein, the term genome editing generally refersto the process of editing or changing the nucleotide sequence of agenome, preferably in a precise or predetermined manner. Examples ofmethods of genome editing described herein include methods of usingsite-directed nucleases to cut genomic DNA at a precise target locationor sequence within a genome, thereby creating a DNA break (e.g., a DSB)within the target sequence, and repairing the DNA break such that thenucleotide sequence of the repaired genome has been changed at or nearthe site of the DNA break.

Double-strand DNA breaks (DSBs) can be and regularly are repaired bynatural, endogenous cellular processes such as homology-directed repair(HDR) and non-homologous end-joining (NHEJ) (see e.g., Cox et al.,(2015) Nature Medicine 21(2):121-131).

DNA repair by HDR utilizes a polynucleotide (often referred to as a“repair template” or “donor template”) with a nucleotide sequence thatis homologous to the sequences flanking the DSB. DNA repair by HDRmechanisms involves homologous recombination between the repair templateand the cut genomic DNA molecule. Repair templates may be designed suchthat they insert or delete nucleotides in the genomic DNA molecule orchange the nucleotide sequence of the genomic DNA molecule.

NHEJ mechanisms can repair a DSB by directly joining or ligatingtogether the DNA ends that result from the DSB. Repair of a DSB by NHEJcan involve the random insertion or deletion of one or more nucleotides(i.e. indels). This aspect of DNA repair by NHEJ is often leveraged ingenome editing methods to disrupt gene expression. NHEJ can also repaira DSB by insertion of an exogenous polynucleotide into the cut site in ahomology-independent manner.

A third repair mechanism is microhomology-mediated end joining (MMEJ),also referred to as “alternative NHEJ”, in which the genetic outcome issimilar to NHEJ in that small deletions and insertions can occur at thecleavage site. MMEJ makes use of homologous sequences of a few basepairsflanking the DNA break site to drive a more favored DNA end joiningrepair outcome (see e.g., Cho and Greenberg, (2015) Nature 518,174-176); Mateos-Gomez et al., Nature 518, 254-57 (2015); Ceccaldi etal., Nature 528, 258-62 (2015). In some instances it may be possible topredict likely repair outcomes based on analysis of potentialmicrohomologies at the site of the DNA break. Each of the aforementionedDNA repair mechanisms can be used in genome editing methods to createdesired genomic alterations. The first step in the genome editingprocess is to create typically one or two DNA breaks in a targetsequence as close as possible to the site of intended mutation oralteration. This can achieved via the use of a site-directed nuclease,as described and illustrated herein.

Hemoglobinopathy: As used herein, the term “hemoglobinopathy” refers toany defect in the structure, function, or expression of any hemoglobinof an individual, and includes defects in the primary, secondary,tertiary or quaternary structure of hemoglobin caused by any mutation,such as deletion mutations or substitution mutations in the codingregions of the β-globin gene, or mutations in, or deletions of, thepromoters or enhancers of such genes that cause a reduction in theamount of hemoglobin produced as compared to a normal condition. Theterm further comprises any decrease in the amount or effectiveness ofhemoglobin, whether normal or abnormal, caused by external factors suchas disease, chemotherapy, toxins, poisons, or the like.B-hemoglobinopathies contemplated herein include, but are not limitedto, sickle cell disease (SCD, also referred to as a sickle cell anemiaor SCA), sickle cell trait, hemoglobin C disease, hemoglobin C trait,hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease,thalassemais, hemoglobins with increased oxygen affinity, hemoglobinswith decreased oxygen affinity, unstable hemoglobin disease, andmethemoglobinemia.

In need: As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment.

Insertion: As used herein, an “insertion” or an “addition” refers to achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acid residues or nucleotides, respectively, to amolecule as compared to a reference sequence, for example, the sequencefound in a naturally-occurring molecule.

Intron: As used herein, the term “intron” refers to any nucleotidesequence within a gene that is removed by RNA splicing mechanisms duringmaturation of the final RNA product (e.g., an mRNA). An intron refers toboth the DNA sequence within a gene and the corresponding sequence in aRNA transcript (e.g., a pre-mRNA). Sequences that are joined together inthe final mature RNA after RNA splicing are “exons”. As used herein, theterm “intronic sequence” refers to a nucleotide sequence comprising anintron or a portion of an intron. Introns are found in the genes of mosteukaryotic organisms and can be located in a wide range of genes,including those that generate proteins, ribosomal RNA (rRNA), andtransfer RNA (tRNA). When proteins are generated from intron-containinggenes, RNA splicing takes place as part of the RNA processing pathwaythat follows transcription and precedes translation.

Lipid: As used herein, the term “lipid” refers to a small molecule thathas hydrophobic or amphiphilic properties. Lipids may be naturallyoccurring or synthetic. Examples of classes of lipids include, but arenot limited to, fats, waxes, sterol-containing metabolites, vitamins,fatty acids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, and polyketides, and prenol lipids. In some instances,the amphiphilic properties of some lipids leads them to form liposomes,vesicles, or membranes in aqueous media.

Modified: As used herein “modified” or “modification” refers to achanged state or change in structure resulting from a modification of apolynucleotide, e.g., DNA. Polynucleotides may be modified in variousways including chemically, structurally, and/or functionally. Forexample, the DNA molecules of the present disclosure may be modified bythe incorporation of a chemically-modified base that provides abiological activity. In one embodiment, the DNA is modified by theintroduction of non-natural or chemically-modified bases, nucleosidesand/or nucleotides, e.g., as it relates to the natural nucleobasesadenine (A), guanine (G), cytosine (C), and thymine (T).

mRNA: As used herein, an “mRNA” refers to a messenger ribonucleic acid.An mRNA may be naturally or non-naturally occurring or synthetic. Forexample, an mRNA may include modified and/or non-naturally occurringcomponents such as one or more nucleobases, nucleosides, nucleotides, orlinkers. An mRNA may include a cap structure, a 5′ transcript leader, a5′ untranslated region, an initiator codon, an open reading frame, astop codon, a chain terminating nucleoside, a stem-loop, a hairpin, apolyA sequence, a polyadenylation signal, and/or one or morecis-regulatory elements. An mRNA may have a nucleotide sequence encodinga polypeptide. Translation of an mRNA, for example, in vivo translationof an mRNA inside a mammalian cell, may produce a polypeptide.Traditionally, the basic components of a natural mRNA molecule includeat least a coding region, a 5′-untranslated region (5′-UTR), a 3′UTR, a5′ cap and a polyA sequence.

Naturally occurring: As used herein, the term “naturally occurring” asapplied to an object refers to the fact that an object can be found innature. For example, a polypeptide or polynucleotide sequence (e.g., asplice site), or components thereof such as amino acids or nucleotides,that is present in an organism (including viruses) that can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

Non-homologous end joining: As used herein, the term “non-homologous endjoining” refers to a pathway that repairs double-strand breaks (DSBs) inDNA. NHEJ is referred to as “non-homologous” because the DNA termini aredirectly ligated without the need for a homologous template, in contrastto homology directed repair (HDR), which requires a homologous sequenceto guide repair.

Non-replicative: As used herein, the term “non-replicative” refers tothe characteristic of a DNA molecule as being unable to replicate withina cell or an organism. Certain DNA molecules (e.g., plasmids, viralgenomes) contain sequence elements (e.g., origins of replications) thatimpart the DNA molecule with the ability to be copied, or replicated, bya cell or organism. The term “non-replicative” connotes those DNAmolecules that do not contain such sequence elements.

Nucleic acid: As used herein, the term “nucleic acid” refers todeoxyribonucleotides or ribonucleotides and polymers or oligomersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides that have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Polymers of nucleotides are referred toas “polynucleotides”. Exemplary nucleic acids or polynucleotides of thedisclosure include, but are not limited to, ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents,RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

Polynucleotides used herein can be composed of any polyribonucleotide orpolydeoxyribonucleotide, which can be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotide can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. A polynucleotide can alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases. “Modified nucleosides” include, for example, asinosine and thymine, when the latter is found in or comprises RNA. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

Nucleobase: As used herein, the term “nucleobase” (alternatively“nucleotide base” or “nitrogenous base”) refers to a purine orpyrimidine heterocyclic compound found in nucleic acids, including anyderivatives or analogs of the naturally occurring purines andpyrimidines that confer improved properties (e.g., binding affinity,nuclease resistance, chemical stability) to a nucleic acid or a portionor segment thereof. Adenine, cytosine, guanine, thymine, and uracil arethe primary or canonical nucleobases predominately found in naturalnucleic acids. Other natural, non-natural, non-canonical and/orsynthetic nucleobases, can be incorporated into nucleic acids, such asthose disclosed herein.

Nucleoside/Nucleotide: As used herein, the term “nucleoside” refers to acompound containing a sugar molecule (e.g., a ribose in RNA or adeoxyribose in DNA), or derivative or analog thereof, covalently linkedto a nucleobase (e.g., a purine or pyrimidine), or a derivative oranalog thereof. As used herein, the term “nucleotide” refers to anucleoside covalently linked to a phosphate group. As used herein, theterm “ribonucleoside” refers to a nucleoside that comprise a ribose anda nucleobase (e.g., adenosine (A), cytidine (C), guanosine (G),5-methyluridine (m⁵U), uridine (U), or inosine (I)).

Operably linked: As used herein, a nucleic acid, or fragment or portionthereof, such as a polynucleotide or oligonucleotide is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence, or fragment or portion thereof.

Polynucleotide/oligonucleotide: As used herein, the terms“polynucleotide” and “oligonucleotide” are used interchangeably andrefer to a single-stranded or double-stranded polymer or oligomer ofnucleotides or nucleoside monomers consisting of naturally-occurringbases, sugars and intersugar (backbone) linkages. The terms“polynucleotide” and “oligonucleotide” also includes polymers andoligomers comprising non-naturally occurring bases, sugars andintersugar (backbone) linkages, or portions thereof, which functionsimilarly. Polynucleotides are not limited to any particular length ofnucleotide sequence, as the term “polynucleotides” encompasses polymericforms of nucleotides of any length. Short polynucleotides are typicallyreferred to in the art as “oligonucleotides”. In the context of thepresent disclosure, such modified or substituted polynucleotides andoligonucleotides are often preferred over native forms because themodification increases one or more desirable or beneficial biologicalproperties or activities including, but not limited to, enhancedcellular uptake and/or increased stability in the presence of nucleases.In some embodiments, the agonists of the disclosure comprisepolynucleotides and oligonucleotides that contain at least one region ofmodified nucleotides that confers one or more beneficial properties orincreases biological activity (e.g., increased nuclease resistance,increased uptake into cells, increased duplex stability, increasedbinding affinity to a target polypeptide).

Palindromic sequence: As used herein, the term “palindromic sequence”(alternatively “palindrome”) refers to a sequence of nucleotides that isself-complementary; wherein the sequence of nucleotides in the 5′ to 3′direction is the same as the sequence of nucleotides comprising thecomplementary strand, when read in the 5′ to 3′. For example, thesequence 5′-ACCTAGGT-3′ is a palindromic sequence because itscomplementary sequence, 3′-TGGATCCA-5′, when read in the 5′ to 3′direction, is the same as the original sequence. In contrast, thesequence 5′-AGTGGCTG-3′ is not a palindromic sequence because itscomplementary sequence, 3′-TCACCGAC-5′, when read in the 5′ to 3′direction, is not the same as the original sequence.

Parenteral administration: As used herein, “parenteral administration,”“administered parenterally,” and other grammatically equivalent phrases,refer to modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intranasal, intraocular, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intracerebral, intracranial, intracarotid and intrasternal injection andinfusion.

Percent identity: As used herein, the term “percent identity,” in thecontext of two or more nucleic acid or polypeptide sequences, refers totwo or more sequences or subsequences that have a specified percentageof nucleotides or amino acid residues that are the same, when comparedand aligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the “percent identity” canexist over a region of the sequence being compared, e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared. For sequence comparison, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters. Thepercent identity between two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. The percentidentity between two nucleotide sequences can be determined using theGAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

Pharmaceutically acceptable: As used herein, the term “pharmaceuticallyacceptable” refers to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues, organs, and/or bodilyfluids of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” refers to, and includes, any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The compositions can include apharmaceutically acceptable salt, e.g., an acid addition salt or a baseaddition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).

Polypeptide: As used herein, the terms “polypeptide,” “peptide”, and“protein” are used interchangeably to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymer.

Preventing: As used herein, the term “preventing” or “prevent” when usedin relation to a condition, refers to administration of a compositionwhich reduces the frequency of, or delays the onset of, symptoms of amedical condition in a subject relative to a subject which does notreceive the composition.

Purified: As used herein, the term “purified” or “isolated” as appliedto any of the proteins (antibodies or fragments) described herein refersto a polypeptide that has been separated or purified from components(e.g., proteins or other naturally-occurring biological or organicmolecules) which naturally accompany it, e.g., other proteins, lipids,and nucleic acid in a prokaryote expressing the proteins. Typically, apolypeptide is purified when it constitutes at least 60 (e.g., at least65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the totalprotein in a sample.

Reprogramming: As used herein, the term “reprogramming” refers to aprocess that alters or reverses the differentiation state of adifferentiated cell (e.g., a somatic cell). Stated another way,reprogramming refers to a process of driving the differentiation of acell backwards to a more undifferentiated or more primitive type ofcell. It should be noted that placing many primary cells into culturecan lead to some loss of fully differentiated characteristics. Thus,simply culturing such cells included in the term differentiated cellsdoes not render these cells non-differentiated cells (e.g.,undifferentiated) or pluripotent cells. The transition of adifferentiated cell to pluripotency requires a reprogramming stimulusbeyond the stimuli that lead to partial loss of differentiated characterin culture. Reprogrammed cells also have the characteristic of thecapacity of extending passaging without loss of growth potential,relative to primary cell parents, which generally have capacity for onlya limited number of divisions in culture.

Sense strand: As used herein the term “sense strand” or “coding strand”refers to a segment within double-stranded DNA (e.g., genomic DNA) witha 5′ to 3′ directionality and has the same nucleotide sequence as anmRNA transcribed from the segment. The transcription product is pre-mRNAtranscript, which contains a sequence of nucleotides that is identicalto that of the sense strand, with the exception that uracil will beincorporated into the mRNA at those positions where thymine is locatedin the DNA. The sense strand is complementary to the antisense strand ofDNA, or template strand, which runs from 3′ to 5′.

Site-directed nuclease: As used herein, the term “site-directednuclease” refers to one of several distinct classes of nucleases thatcan be programmed or engineered to recognize a specific target site(i.e., a target nucleotide sequence) in a DNA molecule (e.g., a genomicDNA molecule) and generate a DNA break (e.g., a DSB) within the DNAmolecule at, near or within the specific site. Site-directed nucleasesare useful in genome editing methods, such as those described herein.Site-directed nucleases include, but are not limited to, the zinc fingernucleases (ZFNs), transcription activator-like effector (TALE)nucleases, CRISPR/Cas nucleases (e.g., Cas9), homing endonucleases (alsotermed meganucleases), and other nucleases (see, e.g., Hafez andHausner, Genome 55, 553-69 (2012); Carroll, Ann. Rev. Biochem. 83,409-39 (2014); Gupta and Musunuru, J. Clin. Invest. 124, 4154-61 (2014);and Cox et al., supra. These differ mainly in the way they bind DNA andcreate the targeted, site-specific DNA break. Site-directed nucleasesknown in the art may produce a single-strand break (SSB) or a DSB. Forthe purposes of the present invention, the disclosure's reference to a“site-directed nuclease” refers to those nucleases that produce a DSB.After creation of a DSB, essentially the same natural cellular DNArepair mechanisms of NHEJ or HDR are co-opted to achieve the desiredgenetic modification. Therefore, it is contemplated that genome editingtechnologies or systems using site-directed nucleases can be used toachieve genetic and therapeutic outcomes described herein.

Stem cell: As used herein, the term “stem cell” is used interchangeablywith the term “hematopoietic stem cell” (HSC). Stem cells aredistinguished from other cell types by two important characteristics.First, stem cells are unspecialized cells capable of renewing themselvesthrough cell division, sometimes after periods of inactivity (e.g.,quiescent state). Second, under certain physiologic or experimentalconditions, stem cells can be induced to become tissue- ororgan-specific cells with special functions. In some organs, such as thegut and bone marrow, stem cells regularly divide to repair and replaceworn out or damaged tissues. In other tissues, such as the pancreas andheart, stem cells only differentiate under certain conditions.

The term “HSC” can refer to multipotent stem cell that is capable ofdifferentiating into all blood cells including erythrocytes, leukocytes,and platelets. HSCs are contained not only in the bone marrow, but alsoin umbilical cord blood derived cells.

Stem cell mobilizer: As used herein, the term “stem cell mobilizer” isused interchangeably with the terms “mobilizer of hematopoietic stem orprogenitor cells” or “mobilize” and refers to any agent, whether it is asmall organic molecule, a polypeptide (e.g., a growth factor or colonystimulating factor or an active fragment or mimic thereof), a nucleicacid, a carbohydrate, an antibody, that acts to enhance the migration ofstem cells from the bone marrow into the peripheral blood. A stem cellmobilizer may increase the number of HSCs or hematopoieticprogenitor/precursor cells in the peripheral blood, thus allowing for amore accessible source of stem cells. In some embodiments, a stem cellmobilizer refers to any agent that mobilizes CD34+ stem cells. It isfurther understood that an agent may have stem cell mobilizing activityin addition to one or more other biological activities including, butnot limited to, immunosuppression.

Subject: As used herein, the term “subject” includes any human ornon-human animal. For example, the methods and compositions of thepresent invention can be used to treat a subject with a disorder (e.g.:a genetic disorder). The term “non-human animal” includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,sheep, dog, cow, chickens, amphibians, reptiles, etc.

Therapeutic agent: As used herein, the term “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeutic,diagnostic, and/or prophylactic effect and/or elicits a desiredbiological and/or pharmacological effect.

Therapeutically effective amount: As used herein, the terms“therapeutically effective amount” or “therapeutically effective dose,”or similar terms used herein are intended to mean an amount of an agentthat will elicit the desired biological or medical response, such as,for example, at least partially arresting the condition or disease andits complications in a patient already suffering from the disease (e.g.,an improvement in one or more symptoms of a cancer). Amounts effectivefor this use will depend on the severity of the disorder being treatedand the general state of the patient's own immune system.

Treat: The terms “treat,” “treating,” and “treatment,” as used herein,refer to therapeutic measures described herein. The methods of“treatment” employ administration of a composition of the disclosure toa subject, in need of such treatment, in order to, cure, delay, reducethe severity of, or ameliorate one or more symptoms of the disorder orrecurring disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

Wild-Type SpCas9: The terms “wild-type SpCas9 nuclease” and “wild-typeSpCas9” refer to a polypeptide having the amino acid sequence of SEQ IDNO: 103 that has biochemical and biological activity when combined witha suitable gRNA to form an active CRISPR/Cas endonuclease system.

Wild-Type SaCas9: The terms “wild-type SaCas9 nuclease” and “wild-typeSaCas9” refer to a polypeptide having the amino acid sequence of SEQ IDNO: 104 that has biochemical and biological activity when combined witha suitable gRNA to form an active CRISPR/Cas endonuclease system.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments, described herein. The scope of the present disclosure isnot intended to be limited to the above Description, but rather is asset forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that thedisclosure encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included, andmethods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES Example 1. In Vitro Screen of DNA DSB Repair Modulators forImproved HDR in T Cells

Multiple pathways are used for repair of DNA double stranded breaks(DSBs). The homology directed repair (HDR) pathway uses homologous donorDNA (e.g., a sister chromatid or exogenous donor DNA) for high fidelityrepair. The efficiency of HDR is generally low due to competition withother repair pathways, notably the non-homologous end-joining (NHEJ)pathway. HDR is predominantly active in the S/G2 phases of the cellcycle, whereas NHEJ repair is active in each phase of the cell cycle andis the predominant repair pathway in G1 cells. Thus, HDR efficiency ispoor in non-dividing or slowly dividing cells, for example, long-termrepopulating hematopoietic cells (LT-HSPCs), lung progenitor cells, orhepatic cells. Given that NHEJ repair is error-prone, frequentlyresulting in small nucleotide insertions or deletions (indels) that cancause a frameshift mutation, it is undesirable for generating precisemodification of a gene (i.e., specific nucleotide changes or knock-in ofa gene).

An in vitro assay was conducted to determine the ability of variousinhibitors and enhancers to improve the efficiency of HDR repair inHEK293 T cells. Specifically, a fluorescent reporter-based screeningapproach was developed to identify molecules that enhance gene-editingby HDR. A reporter system was generated in HEK293 T cells comprising agenomic AAVS1 locus that encoded either a blue fluorescent protein (BFP)or a green fluorescent protein (GFP) within the AAVS1 locus. A geneencoding BFP can be converted to a gene encoding GFP by substitution ofcytosine at position 199 to uridine (e.g., a C to U transition atposition 199). By introducing a DSB within the BFP gene using aCRISPR/Cas9 gene-editing system, a homology donor DNA encoding thenucleotide substitution can be used to edit the BFP gene to a GFP geneby HDR repair. Thus, a gene edit that induces a C to U transition atposition 199 results in a change in cellular fluorescence. A change inthe fluorescence of the cell measured by flow cytometry can be used toquantify efficiency of HDR repair. Additionally, the reverse gene editcan be performed for an AAVS1 locus encoding a GFP gene, wherein a GFPcan be converted to BFP by substitution of uridine at position 199 tocytosine.

To create the reporter system, an AAV-based homology donor DNA encodingeither BFP or GFP was created. The AAV-BFP or AAV-GFP donor was flankedby homology arms that were 1000 base pairs in length and expression ofBFP or GFP was driven by the MND promoter (e.g., a synthetic promoterthat contains the U3 region of a modified Moloney murine leukemia viruslong term terminal repeat with myeloproliferative sarcoma virus enhancerand deleted negative control region, SEQ ID NO: 58). The viral DNA waspackaged into capsids of adeno-associated virus serotype 6 (AAV6)vector. Either the BFP or GFP donor was introduced using the AAV6 vectorinto the AAVS1 locus in HEK293 T cells by homologous recombination usingCRISPR-Cas9 as follows: purified Cas9 protein was complexed with twodifferent single gRNAs (sgRNAs) targeting the AAVS1 locus at SEQ ID NO:3 (spacer sequence identified by SEQ ID NO: 4; gRNA obtained fromMaxcyte) or SEQ ID NO: 5 (spacer sequence identified by SEQ ID NO: 6;gRNA obtained from Thermo Fisher). Cas9 protein used in the Exemplarysection provided herein refers to Cas9 polypeptide derived from S.pyogenes (SpCas9), unless indicated otherwise.

The Cas9-sgRNA complex was electroporated into the HEK293 T cells usingthe Lonza Nucleofector program CM-130. Approximately, 2 hours afterelectroporation, the cells were infected with various viral doses ofAAV6 encoding a BFP (SEQ ID NO: 44) or GFP homology donor (SEQ ID NO:47). One week later, cells were analyzed by flow cytometry to verifyintegration and expression of BFP or GFP in the AAVS1 locus. HEK293 Tcells expressing BFP or GFP were sorted into single cells in 96-wellplates. After ˜2 weeks of growth, DNA extracted from individual cellswas analyzed for precise integration of the respective BFP or GFP geneinto the AAVS1 locus by long-range PCR. This PCR analysis allowed fordetermination of whether the BFP/GFP gene was integrated in one or bothalleles of the AAVS1 locus.

To introduce a gene edit that converts BFP to GFP, cells wereelectroporated with ribonucleoprotein (RNP) comprised of Cas9 and sgRNAthat targets the BFP gene encoded in the AAVS1 locus. The sgRNA targeteda sequence in the BFP gene identified by SEQ ID NO: 7 (sgRNA spacersequence identified by SEQ ID NO: 8). The sgRNA were prepared using astandard sgRNA cassette specific to SpCas9, as indicated by SEQ ID NO: 2(wherein a, c, g, u represent 2′ O-methyl phosphorothioate nucleotides;s represents phosphorothioate nucleotides; and A, C, G, U, N representcanonical RNA nucleotides). The gRNAs used in the Exemplary sectionprovided herein were sgRNAs prepared with the SpCas9 sgRNA cassetteunless indicated otherwise.

The cells were also transfected with single-strandedoligodeoxynucleotide (ssODN) that encoded the gene correction necessaryto convert BFP to GFP and homology arms complimentary to the sequenceupstream and downstream of the target gene cut site. The efficiency ofHDR repair was determined by measuring the level of cell GFPfluorescence.

Molecules that manipulate targets in DSB repair pathways were evaluatedand are listed in Table 2. These included molecules that inhibit targetsthat facilitate repair by the NHEJ pathway, including i53 (polypeptideinhibitor of 53BP1), Nu7441 (DNA-PKcs inhibitor), SCR7 (DNA Ligase IVinhibitor), CYREN1 (Ku70/80 inhibitor), and CYREN2 (Ku70/80 inhibitor).Also evaluated were molecules that enhance repair by the HDR pathway,including RS-1 (Rad51 agonist). Additionally, molecules were evaluatedthat inhibit repair by the alternative end joining (A-EJ) pathway,including siRNA targeting DNA polymerase θ and veliparib (PARPinhibitor). Also evaluated were molecules that affect cell cycle,including XL 413 (CDC7 inhibitor). Finally, also tested was the βadrenergic receptor agonist L755,507 that was previously reported toimprove HDR efficiency (Yu, C. et al. (2015) Cell Stem Cell 16:142-147).

TABLE 2 Pathway Molecule Target Reference NHEJ i53 53BP1 SEQ ID NO: 70NHEJ Nu7441 DNA-PKcs CAS 503468-95-9; Tocris 3712 NHEJ SCR7 DNA LigaseCAS 533426-72-0; Stemcell IV 74102 NHEJ CYREN1 Ku70/80 Arnoult et alNature (2017) and 2 549: 548-552 HDR RS-1 Rad51 CAS 312756-74-4 SigmaR9782 MMEJ siRNA Pol θ MMEJ Veliparib PARP CAS 912444-00-9 Santa Cruz394457 Unknown L755,507 β3-adrenergic CAS 159182-43-1 Tocris 2197receptor Cell Cycle XL 413 CDC7 CAS 1169562-71-3 Tocris 5493

Shown in FIG. 1A is a comparison of HDR editing efficiency for cellstreated with Nu7441, SCR7, or RS-1. The cells were transfected with fourdifferent ssODNs. 2×10⁵ cells were used per reaction, and treated with100 μM of donor DNA. These included ssODN1 (SEQ ID NO: 21), ssODN2 (SEQID NO: 22) and ssODN4 (SEQ ID NO: 24) that are complimentary to the DNAstrand containing the a PAM sequence and ssODN3 (SEQ ID NO: 23) that iscomplimentary to the DNA strand not containing the PAM sequence. Aconcentrated stock solution of each inhibitor was prepared in DMSO anddiluted to a final concentration of 2.5 μM Nu7441, 1.25 μM SCR7, or 10μM RS-1. HDR efficiency was compared relative to treatment with DMSOalone or to treatment with RNP-only. The cells were treated with RNPcontaining 1 μM Cas9 and 1.5 μM sgRNA.

As shown, no improvement in HDR efficiency was seen with treatment ofSCR7 or RS-1 for any of the ssODN constructs tested. However, treatmentwith Nu7441 resulted in approximately 3-fold higher HDR efficiency foreach of the ssODN constructs tested. The improvement in HDR efficiencyfor treatment with Nu7441 was dose-dependent as shown in FIG. 1B. Cellstreated with high concentration of Nu7441 (2.5 μM) had an approximately1.5-fold higher HDR efficiency than cells treated with lowconcentrations of Nu7441 (0.6 μM). This improvement with higher dose wasseen for cells transfected with either ssODN 91-36 (SEQ ID NO: 26) orssODN 91-61 (SEQ ID NO: 27) (FIG. 1C). In each case, treatment withconcentrations of Nu7441 higher than 2.5 μM resulted in no furtherimprovement in HDR efficiency.

A protein inhibitor of 53BP1 was also evaluated for improved HDRefficiency. The choice of repair pathway for repair of a DNA DSB islargely controlled by an antagonism between p53-binding protein 1(53BP1), a pro-NHEJ factor, and BRCA1, a pro-HDR factor (Chapman, J. etal. (2012) Molecular cell 47:497-510). 53BP1 promotes NHEJ repair overHDR repair by suppressing formation of 3′ single-stranded DNA tails,which is the rate-limiting step in the initiation of the HDR pathway.Loss of 53BP1 has been shown to increase HDR efficiency, (Canny, M. etal. (2018) Nat Biotechnol. 36(1):95-102). Thus, inhibition of 53BP1 isexpected to reduce DSB repair by the NHEJ pathway and favor repair bythe HDR pathway. An inhibitor of 53BP1 is the i53 polypeptide (SEQ IDNO: 70). Using the same in vitro assay assessing increased HDR by a BFPto GFP gene conversion, the effect of inhibition of the i53 polypeptideinhibitor (SEQ ID NO: 70) was evaluated. HEK293 T cells were transfectedwith two different ssODNs homologous to the AAVS1 locus: Hn-91-61 (SEQID NO: 25) and Ht-39-88 (SEQ ID NO: 28). 2×10⁵ cells were edited with 1μM Cas9 and 1.5 μM sgRNA, and with 100 μM ssODN. Cells were treated withdifferent doses of an mRNA encoding the i53 polypeptide mRNA openreading frame encoding i53 polypeptide identified by SEQ ID NO: 69). HDRefficiency increased with treatment of mRNA encoding i53 as shown inFIG. 1D.

Additional molecules were tested for improved HDR efficiency byassessing a BFP to GFP gene edit that had no effect. Cells were treatedwith different concentrations of veliparib using ssODN identified by SEQID NO: 25, but no improvement in HDR efficiency was seen as shown inFIG. 1B. Cells were treated with different concentrations of L755,507using either 91-36 ssODN (SEQ ID NO: 26) or 91-61 ssODN (SEQ ID NO: 27),but no improvement in HDR efficiency was seen as shown in FIG. 1C. Cellswere treated with siRNA targeting DNA polymerase θ (Pol θ). However, noimprovement in HDR efficiency was seen for any siRNA dose tested.

Example 2. Increased HDR and Decreased Indel Formation with Treatment byDNA PKcs Inhibitor Nu7441

The effect of DNA PK inhibition by Nu7441 correction of a DSB by theNHEJ pathway (e.g., introduce an indel at the DSB site) or HDR pathway(e.g., introduce a gene mutation encoded by a homology donor at the DSBsite) was assessed using the reporter system described in Example 1. Inthis case gene-editing was evaluated in HEK293 T cells expressing GFP inthe AAVS1 locus. To introduce a gene edit that converts GFP to BFP,cells were electroporated with ribonucleoprotein (RNP) comprised of Cas9and gRNA1 (GFP target sequence identified by SEQ ID NO: 9; sgRNA spacersequence identified by SEQ ID NO: 10) or gRNA2 (GFP target sequenceidentified by SEQ ID NO: 11; sgRNA spacer identified by SEQ ID NO: 12)that targets the GFP gene encoded in the AAVS1 locus. The cells werealso transfected with ssODNs that encoded the gene correction necessaryto convert GFP to BFP and homology arms complimentary to the sequenceupstream and downstream of the target gene cut site. The efficiency ofHDR repair was determined by measuring the level of cell BFPfluorescence. The efficiency of cutting (indel information) wasmonitored by TIDE analysis.

Shown in FIGS. 2A-2B is a comparison of HDR editing efficiency for cellstreated with Nu7441, SCR7, or RS-1. Shown in FIG. 2A are cells wereelectroporated with RNP comprising Cas9 and gRNA1 (GFP target sequenceshown in SEQ ID NO: 9; sgRNA spacer sequence identified by SEQ ID NO:10). 2×10⁵ cells were edited with 1 μM Cas9 and 1.5 μM gRNA1. The cellswere transfected with four different ssODNs. These included ssODN 1067(SEQ ID NO: 29) and ssODN 1069 (SEQ ID NO: 31) that are complimentary tothe DNA strand containing the PAM sequence and ssODN 1068 (SEQ ID NO:30) and ssODN 1070 (SEQ ID NO: 32) that are complimentary to the DNAstrand not containing the PAM sequence. Cells were edited with 100 μMssODN. A concentrated stock solution of each inhibitor was prepared inDMSO and diluted to a final concentration of 2.5 μM Nu7441, 1.25 μMSCR7, or 10 μM RS-1. HDR efficiency was compared relative to treatmentwith DMSO alone or to treatment with RNP-only. As shown, no improvementin HDR efficiency was seen with treatment of SCR7 or RS-1 for any of thessODN constructs tested. However, treatment with Nu7441 resulted inimproved HDR efficiency for each of the ssODN constructs tested FIG. 2A.

Shown in FIG. 2B are cells were electroporated with RNP comprising Cas9and gRNA2 (GFP target sequence shown in SEQ ID NO: 11; sgRNA spacersequence shown in SEQ ID NO: 12). The cells were transfected with fourdifferent ssODNs. These included ssODN 1061 (SEQ ID NO: 33) and ssODN1063 (SEQ ID NO: 35) that are complimentary to the DNA strand containingthe PAM sequence and ssODN 1062 (SEQ ID NO: 34) and ssODN 1064 (SEQ IDNO: 36) that are complimentary to the DNA strand not containing the PAMsequence. Similar to above, treatment with 2.5 μM Nu7441 resulted inimproved HDR efficiency for each of the ssODN constructs tested FIG. 2A.

Shown in FIGS. 2C-2D is a measure of indel formation performed by TIDEanalysis for the same edits that are described for FIGS. 2A-2B.Regardless of the ssODN or gRNA used, treatment with Nu7441 resulted indecreased indel formation at the DSB site, while treatment with SCR7 orRS-1 resulted in reduction compared to a DMSO control. Thus, the DNA PKinhibitor Nu7441 achieves reduced repair of a DSB by the NHEJ pathwaywhen a homology donor is provided.

Having demonstrated improved HDR efficiency for a gene-edit in the AAV1locus upon treatment with Nu7441, its effect on HDR for gene-editing atan additional gene locus was evaluated. HEK293 T cells wereelectroporated with RNP comprised of Cas9 and a gRNA targeting asequence in the GSD1a locus shown in SEQ ID NO: 13 (spacer sequenceidentified by SEQ ID NO: 14). 2×10⁵ cells were edited with 1 μM Cas9 and1.5 μM gRNA. The cells were transfected with two different ssODNshomology donors: 93-50 (SEQ ID NO: 39) or 25-100 (SEQ ID NO: 40). Thesetwo ssODN donors contain homology arms spanning both sides of thedouble-stranded break induced by the Cas9-guide and facilitatecorrection of a point mutation in the G6PC gene sequence by HDR. Cellswere edited with 100 μM ssODN. The cells were treated with 2.5 μMNu7441, 1.25 μM SCR7, or 10 μM RS-1 and the effect on HDR efficiency wasevaluated. While treatment with Nu7441 resulted in an approximately1.7-fold increase in HDR efficiency over DMSO alone, treatment with SCR7or RS-1 had no effect (FIG. 3 ).

The effect of Nu7441 treatment on gene correction by the NHEJ pathwaywas also evaluated. To do so, the cells were transfected with twodifferent dsDNA donors: 50-0 (SEQ ID NO: 37) or 150-0 (SEQ ID NO: 38).Cells were edited with 1.5 μg dsDNA donor. These dsDNA donors, lackinghomology arms, introduce a second 3′ splice site into exon 2 at theGSD1a locus when inserted into the cut site induced by the Cas9-guidecomplex by NHEJ repair. The cells were treated with 2.5 μM Nu7441, 1.25μM SCR7, or 10 μM RS-1 and gene correction by NHEJ repair was evaluated.Treatment with Nu7441 resulted in a substantial decrease in genecorrection for either dsDNA donor compared to treatment with DMSO alone,demonstrating that Nu7441 is inhibiting NHEJ repair following aCas9/gRNA-mediated DNA DSB (FIG. 3 ). Treatment with SCR7 or RS-1 had noeffect over DMSO alone.

The effect of Nu7441 treatment on HDR was also evaluated at the CFTRgene locus in HEK293 T cells. To do so cells were electroporated withRNP comprising Cas9 and a gRNA targeting the CFTR gene locus (SEQ ID NO:18; sgRNA target sequence identified by SEQ ID NO: 19). 2×10⁵ cells wereedited with 1 μM Cas9 and 1.5 μM gRNA. The cells were transfected with assODN donor (SEQ ID NO: 42). Cells were edited with 100 μM ssODN. ThessODN contains homology arms spanning both sides of the DSB induced bythe Cas9-guide and is designed to include 3 additional base pairs (GCA)into the CFTR gene to aid detection of HDR. Cells were also treated with5 μM Nu7441. Gene correction was assessed by TIDE analysis. TIDEanalysis uses a pair of PCR reactions and standard capillary sequencingruns to identify mutations induced at the site of a DSB (see e.g.,Brinkman (2014) Nucleic Acids Res 42:e168). The type of mutation inducedat the DSB was indicative of the pathway used to repair the DSB.Formation of an indel comprising either an insertion or a deletion ofbases was considered due to NHEJ repair; a deletion of 2-3 base pairswas considered due to MMEJ repair; while an insertion of 3 base pairswas considered due to HDR repair. Shown in FIGS. 4A-4B are mutationsintroduced at the DSB for cells treated with Nu7441 (FIG. 4B) comparedto a DMSO negative control (FIG. 4A). While indel formation (e.g., +1,0, or −1 base pair) was high in the negative control (FIG. 4A),indicating high levels of NHEJ repair, indel formation was dramaticallyreduced in cells treated with Nu7441 (FIG. 4B). Additionally, cellstreated with Nu7441 had much higher levels of HDR repair (+3 base pairinsertion) at the DSB. This reduction in NHEJ repair in the presence ofNu7441 was evaluated with an additional ssODN donor (SEQ ID NO: 41). Asshown in FIG. 5 , treatment with either ssODN donor in the presence ofNu7441 resulted in decreased levels of indel formation due to NHEJrepair with a concurrent increase in HDR repair.

Together, these data demonstrate that treatment with Nu7441, a smallmolecule inhibitor of DNA PKcs, results in inhibition of NHEJ repair byCRISPR/Cas9 gene-editing and increased HDR editing efficiency atmultiple gene loci.

Example 3. Efficient Gene Editing by HDR Using i53 at Multiple Gene Lociand in Multiple Cell Types

Having demonstrated improved HDR efficiency with 53BP1 inhibition by thei53 polypeptide, its effect on HDR efficiency at the hemoglobin subunitbeta (e.g., β-globin) (HBB) locus in CD34-expressing LT-HSPCs wasinvestigated.

Frozen CD34-expressing LT-HSPCs derived from plerixafor (i.e.,Mozibil®)+GCSF-dual mobilized peripheral blood obtained from healthyhuman donors were purchased from a commercial vendor. LT-HSPCs weremaintained in culture media comprised of the reagents shown in Table 4and were incubated at 37° C., 5% carbon dioxide, 4% oxygen. The cellswere electroporated with RNP comprised of Cas9 and gRNA targeting theHBB locus (R02 gRNA, target sequence shown in SEQ ID NO: 15). 2×10⁵cells were edited with 3 μg Cas9 and 3 μg gRNA. The target gene sequence(including target sequence with PAM), R02 spacer sequence, and R02 sgRNAsequence are identified in Table 3.

TABLE 3 Sequences of R02 sgRNA SEQ ID Name/Description Sequence NOHBB Target CTTGCCCCACAGGGCAGTAA 15 Sequence HBB TargetCTTGCCCCACAGGGCAGTAACGG 20 Sequence with PAM R02 Spacer SequenceCUUGCCCCACAGGGCAGUAA 16 R02 sgRNA (spacercsususGCCCCACAGGGCAGUAAGUUUUAGAGCUAGAAAUAGCA 17 in bold)AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUGCusususU a, c, g,u: 2′ O-methyl phosphorothioate nucleotides s: phosphorothioatenucleotides A, C, G, U, N: canonical RNA nucleotides

The cells were transfected with a dsDNA homology donor encoding GFPunder a SFFV promoter (SEQ ID NO: 60) that was delivered by AAV (SEQ IDNO: 56). Cells were edited with an AAV dose of 5,000 MOI. The HDRefficiency was determined by measuring the level of GFP fluorescence inthe cells following electroporation. To determine the effect of 53BP1inhibition on HDR efficiency, the 53BP1 inhibitor i53 was introduced asan mRNA-encoded protein to the cells during electroporation withCas9/gRNA RNP and AAV DNA that serves as a donor for homology directedrepair. As shown in FIG. 6A, treatment with mRNA encoding i53polypeptide (SEQ ID NO: 70) resulted in an approximately 1.3-foldincrease in HDR efficiency over cells treated with RNP+AAV-only.Additionally, improved HDR efficiency was seen at each dose of i53 mRNAtested (0.5, 1, and 2 μg mRNA). No GFP expression over background wasseen in cells treated with RNP-only or AAV-only.

TABLE 4 Media components used to culture LT-HSPCs ComponentConcentration Thrombopoietin 100 ng/mL Fms-like tyrosine kinase 3 ligand100 ng/mL Stem cell factor 100 ng/mL Interleukin-3  60 ng/mL

The effect of 53BP1 inhibition on HDR efficiency was compared to othermRNA-encoded proteins that inhibit the NHEJ pathway. These included theproteins CYREN1 and CYREN2 that inhibit Ku70/80, a heterodimer thatbinds DNA blunt ends to prevent processing of 3′ single-stranded DNAtails necessary for HDR repair. Cells were electroporated with RNP, anAAV-GFP homology donor and mRNA encoding either i53 (mRNA ORF shown inSEQ ID NO: 69), CYREN1, or CYREN2. Treatment with i53 mRNA resulted inincreased HDR efficiency when administered at 0.3 μg or 1 μg. Treatmentwith CYREN1 or CYREN2 mRNA resulted in no improvement in HDR efficiencyover RNP+AAV-only (FIG. 6B).

An inhibitor of the cell division cycle7-related (CDC7) protein kinasewas also evaluated for improved HDR editing of the HBB gene locus. CDC7is an initiator of the G1/S transition. Inhibition of CDC7 using thesmall molecule XL413 has been shown to improve HDR efficiency byinducing an early S-phase cell cycle arrest (Wienert, B. et al. (2018)bioRxiv 500462). However, for gene-editing of the HBB locus in LT-HSPCs,no improvement in HDR efficiency was seen for any dose of XL413 tested(data not shown).

The effect of treatment with i53 and Nu7441 was compared for improvingefficiency of HDR repair of the HBB locus. Cells were treated withdifferent doses of Nu7441 or with mRNA encoding i53 (ORF shown by SEQ IDNO: 69). A comparable increase in HDR efficiency was seen upon treatmentwith 0.75 μg of mRNA encoding i53 and 5 μM Nu7441 (FIG. 6C). Noimprovement in HDR efficiency was seen with treatment of a mRNA encodinga non-functional mutant i53 (e.g, DM, mRNA ORF shown by SEQ ID NO: 71).

The effect of treatment of i53 on HDR efficiency was evaluated inadditional cell types, including editing of the AAVS1 locus in humanepithelial cells immortalized with hTERT (hTERT RPE-1, ATCC CRL-4000)cells. RPE1 cells were lipofected with RNP comprised of Cas9 protein andgRNA targeting the AAVS1 locus (target sequence shown in SEQ ID NO: 3;spacer sequence shown in SEQ ID NO: 4). Cells were edited with 1 μg Cas9and 1 μg gRNA. Cells were infected with AAV containing homology donorDNA encoding GFP (DJ serotype) as well as mRNA encoding i53 (mRNA ORFshown in SEQ ID NO: 69). Cells were edited with an AAV dose of 25,000MOI. HDR efficiency was determined by measuring the level of GFPfluorescence in the cells following gene-editing. Treatment of cellswith AAV and mRNA encoding i53 results in an increase in HDR efficiencyover treatment with AAV alone (FIG. 7 ).

Example 4. Efficient Gene Editing of the Hemoglobin Beta Subunit (HBB)Locus in CD34-Expressing LT-HSPCs Using i53 In Vitro

Improved HDR efficiency for CRISPR/Cas9 gene-editing of the HBB locuswith i53 treatment in CD34-expressing LT-HSPCs was evaluated with donorDNA encoding a sickle cell mutation. A sickle mutation is a change of aGAG codon encoding Glu at position 7 of the beta-globin protein to a GUGcodon encoding Val (codon 7 of the HBB open reading frame; E7Vmutation). FIG. 8 shows editing of the HBB locus using a Cas9/gRNAcomplex to introduce a site-specific DSB into exon 1. The homology donorDNA provided introduces a gene correction into exon 1 when repair of theDSB occurs by the HDR pathway. For wild type cells, a homology donor DNAencoding a sickle cell mutation (E7V) can be provided to introduce thesickle mutation into the HBB gene. For cells with the sickle cellmutation, a homology donor DNA encoding a sickle cell correction (E7)can be provided to introduce a sickle correction to the HBB gene.

LT-HSPCs were maintained in culture and gene-editing was performedfollowing two days of culture. Cells were electroporated with RNPcomprised of Cas9 and gRNA targeting the HBB locus (R02 gRNA, targetsequence shown by SEQ ID NO: 15). 1×10⁶ were edited per reaction using20 μg Cas9 and 20 μg gRNA. The cells were electroporated with 1 μg mRNAencoding i53. Electroporation was performed using the Maxcyte HSC-3program. AAV encoding homology donor DNA (AAV.307) was administeredprior to electroporation (pre-EP). Cells were edited with an AAV dose of10,000 MOI. The donor DNA comprised homology arms to the HBB locus andencoded a sickle cell mutation (SEQ ID NO: 53). FIG. 9 shows thesequence of the HBB gene in the region of the gene edit as well as thesickle cell mutation that is introduced following gene editing. Thedownstream PAM recognition site on the HBB locus is indicated, as wellas the sequence recognized by the R02 gRNA spacer. Additionally, aportion of the sequence of the AAV.307 homology donor DNA is shown,including gene changes that are incorporated into the HBB locus by HDRediting. The homology donor incorporates an edit to the PAM sequence toprevent re-cutting of the HBB locus by Cas9/gRNA following editing byHDR. Sequences of the AAV.307 donor are provided in Table 5.

TABLE 5 Sequence of AAV.307 Homology Donor Name/Description SEQ ID NO 5′ITR 112 Left Homology Arm (LHA) 52 Gene-edit (E7 → E7V) 53 RightHomology Arm (RHA) 54 3′ ITR 107 LHA to RHA 110 AAV.307 111

HDR efficiency at the HBB locus was evaluated upon treatment with i53.For assessing frequency of E7V modification of the HBB allele in samplesof edited cells, a next-generating sequencing (NGS) assay using threePCR reactions was performed

Cells were treated with AAV (e.g., AAV.307) and RNP (e.g., Cas9/R02gRNA) and treated with 1 μg of mRNA encoding the i53 polypeptide (SEQ IDNO: 70). Treatment with i53 resulted in 68% incorporation of the E7Vgene edit, an increase of 1.4-fold over RNP+AAV alone (FIG. 10 ).

Editing efficiency at the HBB locus was evaluated by measuring indelformation by TIDE analysis. Electroporation of CD34-expressing LT-HSPCswith RNP comprised of Cas9 and R02 gRNA resulted in 94% indel formation,demonstrating the Cas9/gRNA yields high cutting efficiency within thedesired target gene (FIG. 11 ). Notably, the level of indel formationdecreased for cells treated with RNP+AAV. The lowest level of indelformation was seen for cells treated with RNP+AAV in the presence ofi53, indicating that as the repair pathway shifts towards HDR, indelformation by the NHEJ pathway decreases. This group had 1.7-foldreduction in INDEL frequency relative to cells treated with RNP+AAV,

The effect of i53 on HDR efficiency was evaluated for CD34-expressingLT-HSPCs isolated from human peripheral blood using differentmobilization methods. HDR efficiency was evaluated for LT-HSPCs isolatedfrom human donors following administration of either Mozobil+GCSF orMozobil-alone and gene-edited with AAV+RNP with or without inclusion ofmRNA encoding the i53 polypeptide (SEQ ID NO: 70). Editing was performedby electroporation with RNP containing 20 μg Cas9 and 20 μg R02 gRNA andhomology donor with a E7V mutation encoded by AAV (AAV.307 or AAV.304comprising a gene-edit identified by SEQ ID NO: 50) at a dose of 10,000MOI. Treatment with i53 resulted in approximately 60% incorporation ofthe sickle cell gene-edit by HDR in cells isolated by Mozobil+GCSF,approximately 1.5-fold increase in HDR efficiency over treatment withRNP+AAV alone (FIG. 12A). HDR efficiency in LT-HSPCs isolated usingMozobil and treated with i53 was even higher, with approximately 70% ofcells incorporating the sickle cell gene-edit (FIG. 12B). These resultsdemonstrate that by isolating LT-HSPCs from healthy donors usingmobilization by Mozobil, along with editing in the presence of i53, highlevels of HDR efficiency are achieved.

The growth of CD34+ cells from the time the cells were thawed to rightbefore they were subjected to editing conditions (black bars) wasevaluated and no changes in cell growth were observed. Manipulations ofthe cells by the addition of CRISPR reagents used for editing caused adecrease in the fold growth between when they were thawed and injectedinto mice (blue bars) (FIG. 13 ).

Example 5. Evaluation of LT-HSPCs Edited with i53 Following AdministeredIn Vivo

As described above, HDR efficiency for editing the HBB locus inCD34-expressing LT-HSPCs is improved in the presence of i53. The effectof gene-editing with i53 was evaluated on the ability of the cells toengraft and retain the gene-edit following administration in vivo. Alsoevaluated was the effect of LT-HSPC dose on engraftment followingadministration in vivo.

Human LT-HSPCs were administered to mice following electroporation withCas9/gRNA (R02 gRNA, target sequence identified in SEQ ID NO: 15) andAAV encoding a sickle cell mutation (E7V) as shown in FIG. 14 . Briefly,LT-HSPCs were mobilized from healthy donors using plerixafor. LT-HSPCswere gene-edited following 2 days in culture under conditions describedin Example 3. To perform the gene-edit, cells were electroporated withRNP comprised of Cas9 and gRNA targeting the HBB locus (e.g., R02 gRNA,target sequence SEQ ID NO: 15) and homology donor DNA encoding a sicklecell mutation delivered by AAV (e.g., AAV.307). 1×10⁶ cells were editedwith 20 μg Cas9, 20 μg gRNA and an AAV dose of 10,000 MOI. The cellswere incubated with AAV.307 for 1 hour prior to electroporation. Effectof gene-editing with i53 was determined by treating cells with mRNAencoding i53 polypeptide (SEQ ID NO: 70) during electroporation. Cellswere edited with 1 μg mRNA.

The cells were administered by intravenous injection to cKit mice at 2days following electroporation. Recipient mice were treated withsublethal irradiation (100 cGy) at 1 day prior to administration ofLT-HSPCs to eliminate hematopoietic cells in the bone marrow and enableengraftment of the donor cells. Animals were evaluated for presence ofhuman hematopoietic cells in peripheral blood at 8 and 16 weeksfollowing LT-HSPC administration. The bone marrow was also evaluated forengraftment and maintenance of the HBB gene-edit at 16 weeks followingLT-HSPC administration.

Presence of human hematopoietic cells was measured by flow cytometry inmouse blood or bone marrow samples. The antibodies used for labelingcell-surface markers are shown in Table 6. The gating strategy used toquantify cells by flow cytometry is shown in FIG. 15 . Cells were gatedon singlet, live cells. Mouse and human CD45-expressing hematopoieticcells were distinguished by antibodies targeting mouse or human CD45.Engraftment was measured as percent chimerism which was defined as thequantity of human CD45 positive cells divided by the total number ofCD45 positive cells (e.g., human and mouse CD45 expressing cellscombined). The lineage of human CD45 positive cells was determined usingmarkers for CD19 (e.g., B cells), CD3 (e.g., T cells), CD33 (e.g.,myeloid cells), and CD34 (hematopoietic stem/progenitor cells (HSPCs)).

TABLE 6 Antibody Clone Fluorophore Catalog # Anti-mouse CD45 30-F11 APC103112 Anti-human CD45 HI30 BV786 563716 Anti-human CD19 HIB19 PE-Cy7302216 Anti-human CD3 UCHT1 APC-Cy7 300426 Anti-human CD33 P67.6 PE366608 Anti-human CD34 581 BV421 562577

The effect of titrating the dose of LT-HSPCs on percent chimerism wasevaluated in mouse bone marrow samples collected at 16 weeks followingadministration of LT-HSPCs. Animals received a dose of 0.01×10⁶,0.05×10⁶, 0.1×10⁶, or 0.25×10⁶ LT-HSPCs that were treated withelectroporation, but neither AAV or RNP. As shown in FIG. 16 ,increasing the dose of LT-HSPCs administered to the animals resulted inincreased levels of chimerism. Administration of 0.25×10⁶ LT-HSPCsresulted in approximately 80% human cells within all CD45-expressingcells in the bone marrow. The effect of treatment of i53 on percentchimerism was also evaluated in mouse bone marrow collected at 16 weeksfollowing administration of LT-HSPCs. Animals received a dose of0.25×10⁶ LT-HSPCs that were gene-edited with RNP+AAV either with orwithout i53. Percent chimerism for cells treated with RNP+AAV was lowerthan for cells treated with RNP-only or AAV-only (FIG. 16 ). Inclusionof i53 resulted in no further decrease in chimerism compared to AAV+RNPalone.

The effect of titrating the dose of LT-HSPCs on percent chimerism wasalso evaluated in mouse blood samples collected at 8 and 16 weeksfollowing administration of LT-HSPCs. As seen in the bone marrow,increasing the dose of LT-HSPCs resulted in higher proportion of humancells among CD45-expressing in the blood (FIG. 17 ). Additionally, theproportion of human CD45-expressing cells among total CD45-expressingcells in the blood was lower for LT-HSPCs gene-edited with RNP+AAV aloneor in combination with i53, with approximately 2-3% humanCD45-expressing cells present. These data indicate that cells edited byHDR repair exhibit lower levels of engraftment than unedited cells orcells edited with RNP only.

Shown in FIGS. 18A-18B is lineage analysis of engrafted CD45-expressinghuman cells in mouse bone marrow samples collected at 16 weeks postadministration of LT-HSPCs. The lineage of CD45-expressing humanleukocytes was evaluated for unedited LT-HSPCs administered at a dose of0.01×10⁶, 0.05×10⁶, 0.1×10⁶, or 0.25×10⁶ cells (FIG. 18A). The lineageof CD45-expressing human leukocytes was also evaluated for LT-HSPCsedited with AAV+RNP either in the presence or absence of i53 (FIG. 18B).In both cases no gross changes in lineage distribution were observed forengrafted cells that were edited LT-HSPCs compared to un-editedLT-HSPCs.

Maintenance of gene-editing was evaluated in mouse bone marrow collectedat 16 weeks post-administration of LT-HSPCs. Incorporation of a sicklemutation (E7V) in the HBB locus was evaluated in DNA isolated from mousebone marrow samples using the next generation sequencing assay describedin Example 4. Shown in FIG. 19 is a comparison of HDR efficiency forLT-HSPCs edited either with or without i53. LT-HSPCs were electroporatedwith Cas9/gRNA and treated with AAV. Administration of LT-HSPCs editedwith i53 produced a bone marrow compartment with levels of gene-editingin the HBB locus that were substantially higher than AAV+RNP alone, with65% incorporation of the gene edit by HDR. LT-HSPCs edited in thepresence of i53 had 1.8-fold higher HDR frequency in the bone marrowcompared to cells edited with AAV+RNP alone. These results demonstratethat LT-HSPCs edited with i53 have higher levels of HDR efficiency andthe gene-edit is maintained in cells derived from these progenitor cellsfollowing administration in vivo.

NHEJ editing of the HBB locus was also evaluated in mouse bone marrowcollected at 16 weeks post engraftment. Indel formation at the site ofCas9/gRNA cutting was evaluated by TIDE analysis in bone marrow samplesand compared to indel formation of LT-HSPCs prior to administration.Regardless of the method used to edit the LT-HSPCs, indel formation wassimilar at 16 weeks post-engraftment to the level present prior toadministration, demonstrating persistence of gene-editing followingengraftment of LT-HSPCs (FIG. 20 ). Interestingly, the level of indelformation was lowest for LT-HSPCs gene-edited in the presence of i53,demonstrating that i53 is an effective inhibitor of the NHEJ pathway.

Example 6. Evaluation of Gene Editing of the Hemoglobin Beta Subunit(HBB) Locus in CD34-Expressing LT-HSPCs In Vitro in the Presence of i53or Nu7441

A direct comparison was made of the effect of i53 and Nu7441 on HDRefficiency for CRISPR/Cas9 gene-editing at the HBB locus inCD34-expressing LT-HSPCs using a homology donor DNA encoding a sicklecell mutation. LT-HSPCs were maintained in culture as described inExample 3 and gene-editing was performed following two days in cultureas described in Example 4. Cells were electroporated with RNP comprisedof Cas9 and gRNA targeting the HBB locus (R02 gRNA, target sequenceshown by SEQ ID NO: 15). AAV encoding homology donor DNA (SEQ ID NO: 50)was administered either prior to electroporation (pre-EP) or postelectroporation (post-EP). Additionally, where indicated, cells weretreated with 5 μM Nu7441 or 1 μg of mRNA encoding i53 polypeptide (SEQID NO: 70) during electroporation.

The donor DNA comprised homology arms to the HBB locus and encoded asickle cell mutation. FIG. 21 shows the sequence of the HBB gene in theregion of the gene edit as well as the sickle cell mutation that isintroduced following gene editing. The downstream PAM recognition siteon the HBB locus is indicated, as well as the sequence of the homologydonor DNA (AAV.304), including gene changes that are incorporated intothe HBB locus by HDR editing. The homology donor incorporates an edit tothe PAM sequence to prevent re-cutting of the HBB locus by Cas9/gRNAfollowing editing by HDR. Sequences of the AAV.304 donor are provided inTable 7.

TABLE 7 Sequence of AAV.304 Homology Donor Name/Description SEQ ID NO 5′ITR 112 Left Homology Arm (LHA) 49 Gene-edit (E7 → E7V) 50 RightHomology Arm (RHA) 51 3′ ITR 107 LHA to RHA 108 AAV.304 109

HDR efficiency was evaluated for AAV administered either pre-EP orpost-EP following gene-editing of LT-HSPCs in vitro. For pre-EP, cellswere incubated with AAV for 1 hour prior to electroporation. Forpost-EP, cells were incubated with AAV for 1 hour immediately followingelectroporation. HDR efficiency was evaluated by NGS assay as describedin Example 4. Treatment with RNP and AAV administered either before orafter electroporation resulted in a comparable level of incorporation ofthe gene edit by HDR, approximately 40% (FIG. 22A). Treatment withAAV-only either pre- or post-EP resulted in no incorporation of thedonor DNA in the HBB locus.

HDR efficiency at the HBB locus was evaluated upon treatment with eitheri53 or Nu7441 following gene-editing of LT-HSPCs in vitro. Cells weretreated with pre-EP AAV and RNP and treated with either Nu7441 or mRNAencoding i53. Treatment with Nu7441 resulted in no improvement of HDRefficiency over RNP+AAV alone. However, treatment with i53 resulted in58% incorporation of the E7V gene edit, an increase of 1.4-fold overRNP+AAV alone (FIG. 22B).

Example 7. Evaluation of LT-HSPCs Edited with i53 or Nu7441 FollowingAdministered In Vivo

As described in Example 6, HDR efficiency for editing the HBB locus inCD34-expressing LT-HSPCs is improved in the presence of i53, but not inthe presence of Nu7441. The effect of gene-editing with either i53 orNu7441 was evaluated on the ability of the cells to engraft and retainthe gene-edit following administration in vivo. Also evaluated was theeffect of gene-editing with treatment of AAV prior to electroporation orfollowing electroporation on the ability of the edited cells to engraftand maintain the gene edit following administration in vivo.

Human LT-HSPCs were administered to mice following electroporation withCas9/gRNA and AAV encoding a sickle cell mutation (E7V) as shown in FIG.21 . LT-HSPCs were mobilized from healthy donors using plerixafor.LT-HSPCs were gene-edited following 2 days in culture. To perform thegene-edit, cells were electroporated with RNP comprised of Cas9 and gRNAtargeting the HBB locus (e.g., R02 gRNA, target sequence shown by SEQ IDNO: 15) and AAV encoding a sickle cell mutation (AAV.304). 1×10⁶ cellswere edited with 20 μg Cas9 and 20 μg gRNA. The AAV was administeredeither prior to electroporation (pre-EP) or following electroporation(post-EP). Cells were edited with an AAV dose of 10,000 MOI. Effect ofgene-editing with i53 or Nu7441 was determined by treating cells with 1μg of mRNA encoding i53 polypeptide (SEQ ID NO: 70) or 5 μM Nu7441during electroporation.

A dose of 0.5×10⁶ cells was administered by intravenous injection tocKit mice at 2 days following electroporation. Recipient mice weretreated with sublethal irradiation (100 cGy) at 1 day prior toadministration of LT-HSPCs to eliminate hematopoietic cells in the bonemarrow and enable engraftment of the donor cells. Animals were evaluatedfor presence of human hematopoietic cells in peripheral blood at 8 and16 weeks following LT-HSPC administration. The bone marrow was alsoevaluated for engraftment and maintenance of the HBB gene-edit at 16weeks following LT-HSPC administration.

Percent chimerism was evaluated as described in Example 5 in mouse bloodsamples collected at 8 and 16 weeks post-administration of LT-HSPCs andcompared for LT-HSPCs edited under different conditions. Shown in FIG.23A is a comparison of LT-HSPCs edited with AAV administered eitherpre-EP or post-EP. LT-HSPCs were electroporated with Cas9/gRNA andtreated with AAV prior to electroporation or following electroporation.LT-HSPCs electroporated with RNP demonstrated decreased chimerismrelative to cells that were not electroporated. Treatment with AAVeither pre-EP or post-EP resulted in no improvement in chimerismrelative to treatment with RNP alone. Shown in FIG. 23B is a comparisonof LT-HSPCs edited with pre-EP AAV and RNP in the presence of either i53or Nu7441. Treatment with i53 resulted in levels of chimerism comparableto RNP+AAV alone. However, treatment with Nu7441 resulted in improvedchimerism of approximately 25% at both 8 and 16 weeks, a 2.5-foldincrease over RNP+AAV alone.

Additionally, percent chimerism was evaluated in mouse bone marrowsamples collected at 16 weeks following administration of LT-HSPCs.Shown in FIG. 24A is a comparison of LT-HSPCs edited with AAVadministered either pre-EP or post-EP. Similar to the chimerism seen inmouse blood samples as described above, LT-HSPCs electroporated with RNPdemonstrated decreased chimerism relative to LT-HSPCs that were notelectroporated. Furthermore, treatment with AAV either pre-EP or post-EPresulted in no improvement in chimerism relative to treatment with RNPalone. The chimerism in bone marrow at 16 weeks for LT-HSPCs edited inthe presence of i53 or Nu7441 was also evaluated (FIG. 24B). Treatmentwith i53 resulted in levels of chimerism comparable to RNP+AAV alone.However, treatment with Nu7441 resulted in chimerism that was higherthan RNP+AAV alone and comparable to culture LT-HSPCs that were notelectroporated prior to engraftment. Combined, these results demonstratean unexpected improvement in engraftment for LT-HSPCs gene-edited withtreatment of Nu7441.

Shown in FIG. 25 is lineage analysis of engrafted CD45-expressing humancells in mouse bone marrow samples collected at 16 weeks postadministration of LT-HSPCs. The lineage of CD45-expressing humanleukocytes was compared for LT-HSPCs that were gene-edited in thepresence of i53 or Nu7441. In addition to providing higher levels ofengraftment, treatment with Nu7441 resulted in a greater proportion ofCD34-expressing cells and myeloid cells among human leukocytes in thebone marrow compared to treatment with i53.

Maintenance of gene-editing was evaluated in mouse bone marrow collectedat 16 weeks post-administration of LT-HSPCs. Incorporation of a sicklemutation (E7V) in the HBB locus was evaluated in DNA isolated from mousebone marrow samples using the next generation sequencing assay describedin Example 4. Shown in FIG. 26A is a comparison of HDR efficiency forLT-HSPCs edited with AAV administered either pre-EP or post-EP. LT-HSPCswere electroporated with Cas9/gRNA and treated with AAV prior toelectroporation or following electroporation. Incorporation of thegene-edit in bone marrow samples was similar for LT-HSPCs edited withRNP and AAV given either pre-EP or post-EP. Additionally, incorporationof the gene edit in the HBB locus by HDR was compared for LT-HSPCsedited in the presence of i53 or Nu7441 (FIG. 26B). Administration ofLT-HSPCs edited with Nu7441 produced a bone marrow compartment withlevels of gene-editing in the HBB locus comparable to AAV+RNP alone.However, administration of LT-HSPCs edited with i53 produced levels ofgene-editing in the HBB locus that were substantially higher thanAAV+RNP alone. These results demonstrate that LT-HSPCs edited with i53have higher levels of HDR efficiency and the gene-edit is maintained incells derived from these progenitor cells following administration invivo.

NHEJ editing of the HBB locus was also evaluated in mouse bone marrowcollected at 16 weeks post engraftment. Indel formation at the site ofCas9/gRNA cutting was evaluated by TIDE analysis in bone marrow samplesand compared to indel formation of LT-HSPCs prior to administration.Regardless of the method used to edit the LT-HSPCs, indel formation wassimilar at 16 weeks post-engraftment to the level present prior toadministration (FIG. 27 ). Interestingly, the level of indel formationwas lowest for LT-HSPCs gene-edited in the presence of i53,demonstrating that i53 is an effective inhibitor of the NHEJ pathway.

The functionality of the hematopoietic compartment in recipient mice wasevaluated by measuring erythroid cell enucleation in bone marrowcollected at 16 weeks post-engraftment. Mammalian erythrocytes extrudetheir nucleus prior to entering circulation. Human CD34-expressingLT-HSPCs are expected to differentiate into erythrocytes followingengraftment, however the efficiency of enucleation can be low.Assessment of erythroid cell enucleation provides a measure of theability of edited CD34 expressing cells to differentiate into erythroidcells compared to the unedited controls (i.e., ability to differentiateinto functional cell types). Percent enucleation was compared forLT-HSPCs gene edited with Cas9/gRNA RNP and AAV given pre-EP or post-EP.Levels of enucleation were similar to cells treated with RNP-only orAAV-only (FIG. 28 ). Additionally, levels of enucleation were comparedfor LT-HSPCs gene edited with RNP+AAV in the presence of i53 or Nu7441.Levels of enucleation were also similar to cells treated with RNP-onlyor AAV-only.

Example 8. Evaluation of i53 for In Vitro Correction of Sickle CellMutation in Human Patient-Derived Cells

The effect of i53 on HDR efficiency was evaluated in cells derived frompatients with a sickle cell mutation in the HBB gene. Specifically,CD34-expressing LT-HSPCs derived from human patients with sickle celldisease were edited with SpCas9, R02 guide, and a homology donor DNAencoding a correction to the sickle cell mutation in the HBB gene (i.e.,E6V to E6) delivered by AAV. The AAV-encoded homology donor used forcorrection is referred to as “AAV.323” and is identified by sequence inTable 8. As shown in FIG. 29 , AAV.323 encodes glutamate at position 6of the HBB open reading frame (i.e., E6). However, the codon for E6 is“GAA” rather than wild-type “GAG”, allowing the correction encoded bythe AAV.323 to be detected in wild-type cells or cells encoding the E6Vmutation in the HBB gene.

TABLE 8 Sequence of AAV323 Homology Donor Encoding SCD CorrectionName/Description SEQ ID NO 5′ ITR 106 Left Homology Arm (LHA) 99Gene-edit (E6V → E6) 102 Right Homology Arm (RHA) 100 3′ ITR 107 LHA toRHA 98 AAV.323 105

Briefly, CD34-expressing LT-HSPCs were derived from plerixafor+GCSF-dualmobilized peripheral blood obtained from a human donor with sickle celldisease. The cells were seeded in Phase I media at a cell density of2×10⁵ cells/mL. Cells were cultured at 37° C. under normoxic conditions(i.e., oxygen 20%).

Editing of cells was performed following two days of in vitro culture.Briefly, 5×10⁵ cells were electroporated with RNP containing 20 μgSpCas9 and 20 μg R02 sgRNA, AAV.323 at a dose of 10,000 MOI, and 1 μgmRNA encoding i53. The cells were incubated with AAV.323 for 1 hourprior to electroporation. The cells were edited by electroporation withR02+AAV.323+i53 mRNA and compared to control cells edited with R02 only,R02+AAV.323, or cells exposed to electroporation without RNP or AAVediting components (mock EP).

Following editing, the cells were differentiated to erythrocytes.Briefly, edited cells were plated in fresh Phase I media at a density of2×10⁵ cells/mL, and re-plated at similar density in fresh Phase I mediaon days 3 and 5 post-editing. On day 7 post-editing, the cells wereincubated in Phase II media at a density of 2.5×10⁵ cells/mL. On day 10post-editing, the cells were incubated in Phase III media at a densityof 1.2×10⁶ cells/mL. Cell expansion during culture was monitored overtime and cells electroporated with R02+AAV.323+i53 mRNA grew similarlyto control cells (R02 only, R02+AAV.323, or mock EP cells) (data notshown). Additionally, cell viability was monitored at frequent timepoints beginning day 3 post-editing, and remained greater than 80% foreach treatment group through approximately day 13 of culture.

Efficiency of Gene Edits

The efficiency of gene correction by HDR repair at the HBB gene locuswas evaluated by NGS assay as described in Example 3. Frequency ofINDELs at the R02 cut site was evaluated by NGS analysis. Treatment withi53 resulted in 66% incorporation of the E6V4E6 gene correction, anincrease of 1.4-fold over RNP+AAV.323 alone (FIG. 30A). Additionally,frequency of INDELs at the R02 cut site was 1.9-fold lower for cellsedited in the presence of i53 compared to cells edited with RNP+AAV.323alone (FIG. 30B). Additionally, HDR repair and INDEL formation werecompared at day 0 and day 14 post-editing. As shown in FIGS. 31A-31B,edit incorporated by HDR repair (FIG. 31A) and frequency of INDELs atthe R02 cut site (FIG. 31B) was similar at day 0 and day 14 for eachtreatment group, indicating the edits were retained throughout in vitrodifferentiation to erythrocytes.

Hemoglobin Expression

Hemoglobin expressed by edited cells that were differentiated toerythrocytes was assessed. Hemoglobin A (HbA) is composed of 2alpha-globin and 2 beta-globin units and is the dominant hemoglobin inadult humans. In human carriers of the HBB E6V mutation, a highproportion of total hemoglobin is hemoglobin S (HbS), which is composedof 2 alpha-globin units and 2 beta-globin units with E6V. Thus,proportion of HbS to HbA produced by edited cells was assessed using anHPLC-based quantification to determine if editing resulted in decreasedlevels of hemoglobin associated with sickle cell disease.

Briefly, on day 18 post-editing, 1×10⁶ cells were harvested,centrifuged, and washed with PBS. The cells were prepared for HPLCanalysis. Hemoglobin variants were quantified in cell samples usingreverse-phase HPLC chromatography and gradient elution. As shown in FIG.32A, HbS levels were dramatically reduced and HbA levels increased forcells edited with R02+AAV.332 or R02+AAV.332+i53 compared to mock EPcontrol cells. As shown in FIG. 32B, cells edited in the presence of i53had 66% correction of HBB gene locus by HDR, but a 90% decrease in HbSlevels relative to mock EP control cells. Thus, high levels of HDRachieved with i53 contribute to normalization of hemoglobin expressionproducts.

Erythrocyte Functionality

The ability of edited cells to differentiate to functional erythrocyteswas assessed by determining expression of erythrocyte-associated cellsurface markers and enucleation using flow cytometry on day 18 postediting.

Briefly, 4×10⁵ cells were obtained, and half were stained forerythrocyte cell-surface markers and half were used for detection ofenucleation. For staining cell-surface markers, the cells were incubatedin PBS containing 1% human serum albumin (PBS-A) and an antibodycocktail of anti-CD233(BRIC6-Band3)-FITC, anti-CD71-PE,anti-CD235a(GlyA)-PE/Cy7, and anti-CD49d (α4)-VioBlue. For detection ofenucleation, 2 drops of NucRed nuclear staining reagent was added to 1mL PBS-A, and 100 μL was added to plated cells. Following incubation,both cell samples were labeled with Sytox Blue solution (1:1000 dilutionin PBS-A) for live/dead analysis. Samples were then assessed by flowcytometry. Cells edited with R02 only, R02+AAV.332, or R02+AAV.332+i53each demonstrated levels of enucleation comparable to mock EP controlcells (>30% of cell population having enucleation). Additionally, theproportion of the population that was CD71⁻GlyA⁺ erythrocytes wassimilar for cells edited with R02+AAV.332 or R02+AAV.332+i53 compared tomock EP control cells (>30% of cell population CD71⁻GlyA⁺).

Editing Patient-Derived PBMCs

It was further evaluated if editing of patient-derived PBMCs in thepresence of i53 would yield high levels of correction of the HBB gene.PBMCs were obtained from a human donor with sickle cell disease. ThePBMCs were expanded in StemSpan SFEM II (1×)+StemSpan CC100(1×)+Dexamethasone 1 μM+hEPO 2 IU/mL at 37° C. under normoxic conditions(20% O₂ concentration). The cells were edited following five days of invitro culture. Patient-derived PBMCs were edited with R02, R02+AAV.332,or R02+AAV.332+i53 as described above. On day 8, the cells weretransferred to Phase 1 media, and differentiation to erythrocytes wasperformed through day 18 as described above. Efficiency of HDR at theHBB gene locus was evaluated on day 12 using the NGS assay described inExample 4. Also evaluated was the frequency of INDELs at the R02cut-site as measured by NGS.

As shown in FIG. 33A, the frequency of correction of the HBB gene by HDRrepair in the presence of i53 was approximately 60% in patient-derivedPBMCs. Additionally, the frequency of INDELs was reduced inpatient-derived PBMCs edited in the presence of i53 compared to controlcells (FIG. 33B). The level of HDR repair and the frequency of INDELs inHBB were comparable in PBMCs and CD34-expressing LT-HSPCs edited in thepresence of i53.

Hemoglobin expression was measured by HPLC analysis for edited PBMCs asdescribed above. As shown in FIG. 34 , PBMCs edited in the presence ofi53 had significant reduction in expression of HbS and increasedexpression of HbA compared to mock EP control cells. The ratio of HbS toHbA was comparable for PBMCs edited in the presence of i53 toCD34-expressing LT-HSPCs edited with i53.

Additionally, the functionality of erythrocytes differentiated fromedited PBMCs was evaluated by measuring cell-surface markers using flowcytometry as described above. PBMCs edited with either R02+AAV.323 orR02+AAV.232+i53 had similar levels of CD71⁻GlyA⁺ erythrocytes to controlcells (R02 only or Mock EP cells), indicating edited cells undergoingHDR repair of the HBB locus properly differentiate to matureerythrocytes (data not shown).

Example 9. Analysis of Off-Target Genomic Editing with R02 gRNA

Off-target sites were investigated that hybridize and are edited by theR02 gRNA when provided as an RNP complex with wild-type SpCas9polypeptide. Briefly, an analysis to identify putative off-target siteswas performed using two approaches. The first approach was to screen thehuman genome to identify genomic sequences complementary to the R02spacer sequence with i) up to 3 mismatches, or ii) 2 mismatches and 1gap. The homology computation off-target prediction was performed usingCCTOP, CRISPOR, and COSMID algorithms. Using this approach, 179off-target sequences were predicted to have homology to the R02 spacersequence.

The second approach was to screen candidate off-target sites usingGUIDE-Seq (see, e.g., Tsai et al (2015) NAT. BIOTECHNOL. 33:187). Basedon this approach, 36 sites in the genomic DNA were identified asundergoing off-target CRISPR/Cas9 cleavage using the R02 gRNA.

Candidate off-target sequences were screened using a quantitative hybridcapture assay. Briefly, 5×10⁵ CD34-expressing LT-HSPCs were thawed andelectroporated with RNP containing 15 μg SpCas9 polypeptide and 15 μgR02 sgRNA. Edited cells were treated with RNP containing SpCas9polypeptide obtained from two separate commercial vendors (referred toas WT SpCas9_1 and WT SpCas9_2). Control cells were electroporatedwithout RNP.

Edited and control cells were harvested, and genomic DNA was extractedusing a DNeasy kit (Qiagen). The genomic DNA samples were hybridizedwith short probes that were prepared to overlay the region of thegenomic DNA that included the putative off-target sequences. Boundgenomic DNA was then enriched using a pull-down purification targetingthe hybridization probe. The genomic DNA was then sequenced forfrequency of INDELs by NGS analysis. The ratio of total number of readswith INDELs to the total number of reads was quantified for eachputative target site for genomic DNA isolated from edited cells andcontrol cells. For putative off-target sites with a frequency of INDELsexceeding 0.2% in edited cells compared to control cells, the targetsite was evaluated by statistical testing. A paired, on-sided T test wasused to identify sites with significant difference in frequency ofINDELs between edited and control cells (i.e., p<0.05).

Based on this analysis, two sites were identified as having astatistically significant level of off-target editing with the R02gRNA/Cas9 complex using either WT SpCas9_1 or WT SpCas9_2. The sitesidentified were OT1 located at chr9:101,833,575-101,833,624 and OT2located at chr12:124,319,275-124,319,308, each location relative tohuman reference genome hg38.

It was evaluated whether gene-editing at off-target sites would bereduced by combining R02 with a SpCas9 variant engineered for increasedfidelity, while retaining on-target cutting efficiency.

SpCas9 variants having a R691A mutation have been reported to haveincreased fidelity (i.e., high fidelity or HiFi) by reducing Cas9nuclease activity at sites with gRNA mismatches, while maintainingcutting efficiency at on-target sites (see, e.g., Vakulskas, et al(2018) NAT MED 24:1216). Accordingly, RNP complex containing R02 sgRNAand an SpCas9 variant having a R691A mutant were evaluated for frequencyof edits at on-target and off-target sites in CD34-expressing LT-HSPCs.Specifically, two SpCas9 R691A HiFi variants were evaluated, a firstvariant having an N-terminal and C-terminal sv40 NLS (referred to as HFSpCas9_1) and a second variant having three N-terminal NLS sequences(referred to as HF SpCas9_2).

For editing, 5×10⁵ CD34-expressing LT-HSPCs were electroporated with RNPcontaining 15 μg of R02 sgRNA and 15 μg of either HF_SpCas9_1 orHF_SpCas9_2. Additionally, the cells were electroporated with 10,000 MOIof AAV.307. Control cells were edited under the same conditions usingRNP containing either WT SpCas9_1 or WT SpCas9_2.

The efficiency of gene-editing at the HBB gene by HDR was evaluatedusing the NGS sequencing assay described in Example 4 on day 2post-editing. As shown in FIG. 35A, the level of HDR using either HiFiSpCas9 variant was comparable to the level of HDR for editing usingwild-type SpCas9. The level of HDR for each SpCas9 is shown in Table 9.

The frequency of INDELs at the OT1 and OT2 cut-sites was evaluated usinggenomic DNA extracted from edited cells. Primers flanking the OT1 andOT2 sites were used to amplify regions of the genomic DNA encompassingthese sites by PCR. The PCR products were purified and sequenced usingNGS. The sequencing data was analyzed to determine the frequency ofINDELs at the OT1 and OT2 sites. As shown in FIG. 35B, the frequency ofINDELs at the OT1 site was substantially reduced using either HiFiSpCas9 variant compared to that induced using wild-type SpCas9. As shownin FIG. 35C, frequency of INDELs at the OT2 site was negligible with useof either HiFi SpCas9 variants.

Quantification of INDEL frequency at the OT1 and OT2 for each SpCas9variant is further shown in Table 9.

TABLE 9 On-Target and Off-Target Editing by SpCas9/R02 gRNA RNP SpCas9 %HDR OT1 % INDEL OT2 % INDEL WT SpCas9_1 40.3 48.7 0.7 WT SpCas9_2 43.828.8 0.3 HF SpCas9_1 42.0 1.6 0.1 HF SpCas9_2 42.5 1.2 0.1

Example 10. Analysis of Hemoglobin Monomers in Edited CD34-ExpressingLT-HSPCs

The hemoglobin monomers produced by CD34-expressing LT-HSPCs followingCRISPR/Cas editing with R02 gRNA and subsequent in vitro differentiationwas investigated. Briefly, CD34-expressing LT-HSPCs derived from healthyhuman patient donors and human patient donors with a SCD mutation (E6V)were isolated and seeded in culture as described in Example 8.

Editing of cells was performed following two days of in vitro culture.Briefly, 5×10⁵ cells were electroporated with RNP containing 20 μgSpCas9 and 20 μg R02 sgRNA and 10,000 MOI AAV.323. The cells wereincubated with AAV.323 for 1 hour prior to electroporation. The cellswere edited by electroporation with R02 RNP+AAV.323 or R02 RNP only andcompared to control cells electroporated without RNP or AAV (mock EP).Subsequently, the cells were differentiated by in vitro culture asdescribed in Example 8.

Hemoglobin monomers produced by differentiated cells was assessed on day18 post-editing. Briefly, approximately 1×10⁶ cells were harvested,centrifuged, and prepared for HPLC analysis. Hemoglobin monomersexpressed by edited cells were detected using LC-MS with separation byreverse phase chromatography. The chromatography enabled separation andquantification of hemoglobin variants (e.g., beta-globin, delta-globin,alpha-globin, gamma2-globin, and gamma1-globin). Beta-globin variantswere further differentiated based on elution time. These includedwild-type beta globin (B), beta globin with SCD mutation (S) and unknownbeta-globin monomers (U). The unknown beta-globing monomers were furthercharacterized based upon analysis by mass spectrometry.

Editing with R02 RNP alone induces a high frequency of INDELs in the HBBgene. Such INDELs can introduce frameshift mutations in HBB that disruptgene expression. Thus, LT-HSPCs edited with R02 RNP and differentiatedto erythrocytes are expected to produce decreased levels of beta-globinmonomers (i.e., B+S+U) relative to total hemoglobin. It was evaluated ifediting using R02 RNP+AAV would prevent this phenotype by reducingfrequency of INDELs in the HBB gene.

As shown in FIG. 36A, CD34-expressing LT-HSPCs edited with R02 RNP alonehad an approximately 1.8-fold decrease in beta-globin monomers (B+S+U)relative to total hemoglobin compared to mock EP control cells,indicating overall reduced expression of beta-globin monomers. Incontrast, cells edited with R02 RNP+AAV had no significant differencecompared to mock EP control cells in the level of beta-globin monomers(B+S+U) relative to total hemoglobin.

Furthermore, the level of gamma globin expressed by edited cellsfollowing in vitro differentiation was also assessed using the LCMSassay. As shown in FIG. 36B, the level of total gamma-globin relative tototal hemoglobin was increased in cells edited with either R02 RNP aloneor R02+AAV relative to mock EP control cells. These data indicateupregulation of gamma-globin in edited cells can contribute toproduction of functional hemoglobin (i.e., HbF), potentially off-settingany decrease in beta-globin production.

The beta globin monomers produced by edited cells were compared tosequence reads from NGS analysis of the region surrounding the R02 cutsite. CD34-expressing LT-HSPCs derived from either a patient donor withSCD or a healthy donor were edited with R02 RNP or R02 RNP+AAV asdescribed above. INDELs induced at the R02 cut site were evaluated byNGS analysis, and the sequence reads were used to identify the mostcommon INDELs induced in the HBB gene following editing in both thewild-type HBB gene and the HBB gene with a E6V mutation. Based on thisanalysis, a deletion of 9 nucleotides surrounding the R02 cut site asshown in FIG. 36C was determined as occurring in both LT-HSPCs derivedfrom healthy or SCD patient donors. Indeed, 20-30% of sequence readsfrom the cells edited with R02 RNP and 3-9% of sequence reads from thecells edited with R02 RNP+AAV corresponded to the −9 nucleotide INDEL.

Example 11. Increased HDR Efficiency with DNA-PK Inhibitors for Editingthe HBB Gene in CD34-Expressing LT-HSPCs

Potent inhibitors of the DNA-PK enzyme complex that functions in theNHEJ repair machinery were evaluated for blocking NHEJ repair andimproving HDR efficiency when used with CRISPR/Cas components forediting the HBB gene locus. Specifically, compounds 984 and 296 havebeen reported as reversible inhibitors of the DNA-PK catalytic subunit(DNA-PKcs), for example, see U.S. Pat. No. 9,592,232 which is hereinincorporated by reference. The compounds have high affinity andselectivity for DNA-PK.

Structure of compounds 984 and 296 are provided in Table 10.

TABLE 10 Structures of DNA-PK Inhibitors

Compound 984

Compound 296

The effect of DNA-PK inhibition using Compound 296 was compared to theeffect of 53BP1 inhibition using i53 for increased HDR repair of the HBBgene locus with donor DNA encoding GFP. Specifically, frozenCD34-expressing LT-HSPCs isolated from plerixafor-mobilized orplerixafor/GCSF-dual mobilized peripheral blood obtained from healthyhuman donors were thawed and seeded in media with components asdescribed in Example 3. The cells were maintained in culture, andgene-editing was performed following two days of culture.

For editing, 5×10⁵ CD34-expressing LT-HSPCs were electroporated with RNPcontaining 20 μg SpCas9 and 20 μg R02 gRNA. Electroporation wasperformed using the CA-137 program of the Lonza Amaza™ 4D-Nucleofector™.The cells were electroporated with a dsDNA homology donor encoding GFPunder a SFFV promoter that was delivered by AAV at a dose of 10,000 MOI.The AAV donor was administered 1 hour prior to electroporation. Thecells were electroporated with compound 296 at a concentration rangingfrom 0.014 μM to 10 μM. For comparison, positive control cells wereelectroporated with 1 μg i53 mRNA and negative control cells wereelectroporated with RNP+AAV only.

The efficiency of HDR for repair of the HBB gene with donor encoding GFPwas determined by measuring the percentage of cells expressing GFPfollowing electroporation. Edited cells were evaluated for GFPfluorescence on day 2 post-editing using flow cytometry, and viabilitywas determined via staining with Tryphan Blue.

As shown in FIG. 37A, the level of HDR repair for cells edited withtreatment of 1.1 μM, 3.3 μM, or 10 μM of compound 296 (53.5%, 56.5%, and55.9% respectively) was significantly higher than for negative controlcells edited without compound 296 (44.1%). Additionally, viability ofedited cells was high for each concentration of compound 296 evaluated(>80%). As shown in FIG. 37B, the level of HDR repair for cells editedwith treatment of 1.1 μM, 3.3 μM, or 10 μM of compound 296 wascomparable to cells edited with i53 mRNA.

The DNA-PK inhibitors were evaluated in combination with R02 RNP andAAV.307 for increasing HDR efficiency at the HBB gene locus, andcompared to effect of editing with i53. Editing was performed asdescribed above. However, the cells were electroporated with AAV.307administered 1 hour prior to electroporation at a dose of 10,000 MOI.The cells were electroporated with compound 296 at a concentrationranging from 0.005 μM to 10 μM, with compound 984 at a concentrationranging from 0.005 μM to 10 μM, or with 1 μg i53 mRNA. Negative controlcells were electroporated with RNP+AAV only or RNP only.

Edited cells were evaluated for viability as described above, and forincorporation of gene-edits at 2 days post-electroporation. Theefficiency of HDR repair for insertion of an E6V mutation in the HBBgene was quantified by NGS assay as described in Example 4. Thefrequency of INDELs induced at the R02 cut site was also evaluated byNGS analysis.

As shown in FIGS. 38A-38B, the level of HDR repair for cells edited with0.37-10 μM of compound 296 was 70% or higher. Indeed, treatment with 3.3μM of compound 296 resulted in editing with 80% HDR efficiency. HDRefficiency at these concentrations was at least 1.6-fold higher than forcontrol cells edited with R02 RNP+AAV only. Moreover, the frequency ofINDELs at these concentrations was reduced by at least 4.6 fold comparedto control cells edited with R02 RNP only.

The level of HDR repair for cells edited with i53 was also high (59%),with reduced frequency of INDELs relative to control cells. In anindependent experiment testing equivalent conditions (“Experiment 2”),levels of HDR and INDEL frequency were similar for each of theconcentrations of compound 296 evaluated (FIG. 38C). Moreover, viabilityof edited cells was high at each concentration of compound 296 used forediting (>80%).

The INDELs species identified for cells edited in Experiment 2 werefurther evaluated to determine frequency of repair by NHEJ or MMEJrepair pathways. An INDEL of ±1 nt was considered due to NHEJ repair; adeletion of −9 nt was considered due to MMEJ repair based on themicrohomology present on either side of the R02 cut site. Based onpercentage of total reads corresponding to these INDEL species, theratio of gene edits due to NHEJ and MMEJ repair was evaluated. As shownin FIG. 38D, cells edited in the presence of compound 296 had up to a30-fold decrease in INDELs due to NHEJ repair, with only a modestreduction in INDEL species due to MMEJ repair. Thus, reduced frequencyof INDELs with treatment of compound 296 is largely due to suppressionof the NHEJ repair pathway.

Evaluation of cells edited with compound 984 also demonstrated highlevels of HDR editing and decreased frequency of INDELs in the HBB gene.As shown in FIGS. 39A-39B, the level of HDR repair for cells edited withtreatment of 0.37-10 μM of compound 984 was high, with treatment of 3.3μM or 10 μM in particular resulting in >80% HDR efficiency compared to54% HDR efficiency in cells edited with R02 RNP+AAV only. The frequencyof INDELs at these concentrations were also significantly reducedcompared to control cells edited with R02 RNP only (i.e., byapproximately 8-fold). Additionally, good viability was observed forcells following editing with compound 984, particularly at minimalconcentrations that were identified as effective for editing.

Together these data demonstrate a substantial improvement in HDR editingefficiency and decreased frequency of INDELs in the HBB gene locus whenDNA-PK inhibitors compound 296 or 984 are combined with RNP containingR02 gRNA.

Sequence Listing SEQ Name/ ID NO: Identifier Description Sequence 1gRNA- sgRNA 1n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaa relatedaaguggcaccgagucggugcu₍₁₋₈₎ 2 gRNA- SpCas9 csususN₍₁₇₋₃₀₎ related sgRNAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCususus U 3 gRNA- AAVS1 targetGGGGCCACTAGGGACAGGAT related sequence A 4 gRNA- AAVS1GGGGCCACUAGGGACAGGAU related sgRNA spacer A 5 gRNA- AAVS1 targetGCCAGTAGCCAGCCCCGTCC related sequence B 6 gRNA- AAVS1GCCAGUAGCCAGCCCCGUCC related sgRNA spacer B 7 gRNA- BFP targetTGAAGCACTGCACGCCAT related sequence 8 gRNA- BFP sgRNA UGAAGCACUGCACGCCAUrelated spacer 9 gRNA- GFP target GCTGAAGCACTGCACGCCGT relatedsequence A 10 gRNA- GFP sgRNA GCUGAAGCACUGCACGCCGU related spacer A 11gRNA- GFP target CTCGTGACCACCCTGACCTA related sequence B 12 gRNA-GFP sgRNA CUCGUGACCACCCUGACCUA related spacer B 13 gRNA- GSD1a targetTCTTTGGACAGCGTCCATAC related sequence 14 gRNA- GSD1a Ch32UCUUUGGACAGCGUCCAUAC related gRNA spacer 15 gRNA- HBB targetCTTGCCCCACAGGGCAGTAA related sequence 16 gRNA- HBB RO2CUUGCCCCACAGGGCAGUAA related sgRNA spacer 17 gRNA- HBB sgRNAcsususGCCCCACAGGGCAGUAAGUUUUAGAGCUAGAAAUAG relatedCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCusususU 18gRNA- CFTR target TCTGTATCTATATTCATCAT related sequence 19 gRNA-CFTR sgRNA UCUGUAUCUAUAUUCAUCAU related spacer 20 gRNA- HBB TargetCTTGCCCCACAGGGCAGTAACGG related Sequence (PAM in bold) 21 DonorssODN1 (Ht- GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC DNA CR282)GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACGTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG A 22 Donor ssODN2 (Hn-TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACG DNA CR283)TCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTG GC 23 Donor ssODN3 (Hn-CGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAG DNA 39-88)TCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACGTCAGGGTGGTCACGAGGGTGGGCCAGGGCAC GG 24 Donor ssODN4 (Ht-GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC DNA 91-61)GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACGTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGC 25Donor ssODN (Hn- GCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAG DNA 91-61)CGGCTGAAGCACTGCACGCCGTACGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGA ACTTCAGGGTCAGCTTGCCGTAGGTGGC26 Donor ssODN (Hn- TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACG DNA91-36) TCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTG GC 27 Donor ssODN (Ht-GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC DNA 91-61)GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACGTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGC 28Donor ssODN (Ht- CGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAG DNA 39-88)TCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACGTCAGGGTGGTCACGAGGGTGGGCCAGGGCAC GG 29 Donor ssODN 1067GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC DNAGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAGCcACGGGGTGCAGTGCTTCAGCCGCTACCCCGACCACATG A 30 Donor ssODN 1068TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACCCCGTGGC DNATCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTG GC 31 Donor ssODN 1069TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC DNATCGTGACCACCCTGAGCcACGGGGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA T 32 Donor ssODN 1070ATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGG DNATAGCGGCTGAAGCACTGCACCCCGTGGCTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGAT GA 33 Donor ssODN 1061CTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTC DNAGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACCCCGTGGCTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGG GC 34 Donor ssODN 1062GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAGCCACGG DNAGGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA G 35 Donor ssODN 1063GACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGG DNACTGAAGCACTGCACCCCGTGGCTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTC A 36 Donor ssODN 1064TGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC DNACCACCCTCGTGACCACCCTGAGCcACGGGGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGT C 37 Donor 50-0 dsDNACCCAGAAACTTGTTCTGTTTTTCCATAGGATTCTCTTTGGACA DNA GTGCCCT 38 Donor150-0 dsDNA AGGGCACTGTCCAAAGAGAATCCTATGGAAAAACAGAACAA DNAGTTTCTGGGGTTACTGAATGAATGCTTTTGCCCAAAGCCTACACCTTCAAGAAGAGTGTAGCCTGAGAAGGATTTCACATGTTG CCTCTAGAAGGGAGAACTGGGTGGC 39Donor 93-50 ssODN TCTTGAAGGTGTAGGCTTTGGGCAAAAGCATTCATTCAGTAA DNACCCCAGAAACTTGTTCTGTTTTTCCATAGGATTCTCTTTGGACAGTGCCCTTACTGGTGGGTCCTGGATACTGACTACTACAGCA ACACTTCCGTGCCCT 40 Donor25-100 TAGGATTCTCTTTGGACAGTGCCCTTACTGGTGGGTCCTGGAT DNA ssODNACTGACTACTACAGCAACACTTCCGTGCCCCTGATAAAGCAGTTCCCTGTAACCTGTGAGACTGGACCAGGTAAGCGTCCCA 41 Donor H3-95-30TAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGG DNA ssODNATTATGCCTGGCACCATTAAAGAAAATATCATAAGCTTTGGTGTTTGCTATGATGAATATAGATACAGAAGCGTCATCAAAG 42 Donor N1-95-30AATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCC DNA ssODNTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTGCTAGCATGATGAATATAGATACAGAAGCGTCATCA 43 Donor AAVS1 locusCCCCAGCTCTTCTCTGTTCAGCCCTAAGAATCCTGGCTCCAGC DNA LHA (used forCCCTCCTACTCTAGCCCCCAACCCCCTAGCCACTAAGGCAAT BFP donor)TGGGGTGCAGGAATGGGGGCAGGGTACCAGCCTCACCAAGTGGTTGATAAACCCACGTGGGGTACCCTAAGAACTTGGGAACAGCCACAGCAGGGGGGCGATGCTTGGGGACCTGCCTGGAGAAGGATGCAGGACGAGAAACACAGCCCCAGGTGGAGAAACTGGCCGGGAATCAAGAGTCACCCAGAGACAGTGACCAACCATCCCTGTTTTCCTAGGACTGAGGGTTTCAGTGCTAAAACTAGGCTGTCCTGGGCAAACAGCATAAGCTGGTCACCCCACACCCAGACCTGACCCAAACCCAGCTCCCCTGCTTCTTGGCCACGTAACCTGAGAAGGGAATCCCTCCTCTCTGAACCCCAGCCCACCCCAATGCTCCAGGCCTCCTGGGATACCCCGAAGAGTGAGTTTGCCAAGCAGTCACCCCACAGTTGGAGGAGAATCCACCCAAAAGGCAGCCTGGTAGACAGGGCTGGGGTGGCCTCTCGTGGGGTCCAGGCCAAGTAGGTGGCCTGGGGCCTCTGGGGGATGCAGGGGAAGGGGGATGCAGGGGAACGGGGATGCAGGGGAACGGGGCTCAGTCTGAAGAGCAGAGCCAGGAACCCCTGTAGGGAAGGGGCAGGAGAGCCAGGGGCATGAGATGGTGGACGAGGAAGGGGGACAGGGAAGCCTGAGCGCCTCTCCTGGGCTTGCCAAGGACTCAAACCCAGAAGCCCAGAGCAGGGCCTTAGGGAAGCGGGACCCTGCTCTGGGCGGAGGAATATGTCCCAGATAGCACTGGGGACTCTTTAAGGAAAGAAGGATGGAGAAAGAGAAAGGGAGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCCTCACCACAGC CCTG 44 Donor BFP locusATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCC DNA donorCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCCATGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAG 45 DonorAAVS1 locus ACTGTGGGGTGGAGGGGACAGATAAAAGTACCCAGAACCAG DNA RHA (used forAGCCACATTAACCGGCCCTGGGAATATAAGGTGGTCCCAGCT BFP donor)CGGGGACACAGGATCCCTGGAGGCAGCAAACATGCTGTCCTGAAGTGGACATAGGGGCCCGGGTTGGAGGAAGAAGACTAGCTGAGCTCTCGGACCCCTGGAAGATGCCATGACAGGGGGCTGGAAGAGCTAGCACAGACTAGAGAGGTAAGGGGGGTAGGGGAGCTGCCCAAATGAAAGGAGTGAGAGGTGACCCGAATCCACAGGAGAACGGGGTGTCCAGGCAAAGAAAGCAAGAGGATGGAGAGGTGGCTAAAGCCAGGGAGACGGGGTACTTTGGGGTTGTCCAGAAAAACGGTGATGATGCAGGCCTACAAGAAGGGGAGGCGGGACGCAAGGGAGACATCCGTCGGAGAAGGCCATCCTAAGAAACGAGAGATGGCACAGGCCCCAGAAGGAGAAGGAAAAGGGAACCCAGCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGCCGAGAAGGAAGTGCTCCGGAAAGAGCATCCTTGGGCAGCAACACAGCAGAGAGCAAGGGGAAGAGGGAGTGGAGGAAGACGGAACCTGAAGGAGGCGGCAGGGAAGGATCTGGGCCAGCCGTAGAGGTGACCCAGGCCACAAGCTGCAGACAGAAAGCGGCACAGGCCCAGGGGAGAGAATGCAGGTCAGAGAAAGCAGGACCTGCCTGGGAAGGGGAAACAGTGGGCCAGAGGCGGCGCAGAAGCCAGTAGAGCTCAAAGTGGTCCGGACTCAGGAGAGAGACGGCAGCGTTAGAGGGCAGAGTTCCGGCGGCACAGCAAGGGCACTCGGGGGCGAGAGGAGGGCAGCGCAAAGTGACAATGGCCAGGGCCAGGCAGATAGA CCAGACTGAGCTATGG 46 DonorAAVS1 locus CCCCAGCTCTTCTCTGTTCAGCCCTAAGAATCCTGGCTCCAGC DNALHA (used for CCCTCCTACTCTAGCCCCCAACCCCCTAGCCACTAAGGCAAT GFP donor)TGGGGTGCAGGAATGGGGGCAGGGTACCAGCCTCACCAAGTGGTTGATAAACCCACGTGGGGTACCCTAAGAACTTGGGAACAGCCACAGCAGGGGGGCGATGCTTGGGGACCTGCCTGGAGAAGGATGCAGGACGAGAAACACAGCCCCAGGTGGAGAAACTGGCCGGGAATCAAGAGTCACCCAGAGACAGTGACCAACCATCCCTGTTTTCCTAGGACTGAGGGTTTCAGTGCTAAAACTAGGCTGTCCTGGGCAAACAGCATAAGCTGGTCACCCCACACCCAGACCTGACCCAAACCCAGCTCCCCTGCTTCTTGGCCACGTAACCTGAGAAGGGAATCCCTCCTCTCTGAACCCCAGCCCACCCCAATGCTCCAGGCCTCCTGGGATACCCCGAAGAGTGAGTTTGCCAAGCAGTCACCCCACAGTTGGAGGAGAATCCACCCAAAAGGCAGCCTGGTAGACAGGGCTGGGGTGGCCTCTCGTGGGGTCCAGGCCAAGTAGGTGGCCTGGGGCCTCTGGGGGATGCAGGGGAAGGGGGATGCAGGGGAACGGGGATGCAGGGGAACGGGGCTCAGTCTGAAGAGCAGAGCCAGGAACCCCTGTAGGGAAGGGGCAGGAGAGCCAGGGGCATGAGATGGTGGACGAGGAAGGGGGACAGGGAAGCCTGAGCGCCTCTCCTGGGCTTGCCAAGGACTCAAACCCAGAAGCCCAGAGCAGGGCCTTAGGGAAGCGGGACCCTGCTCTGGGCGGAGGAATATGTCCCAGATAGCACTGGGGACTCTTTAAGGAAAGAAGGATGGAGAAAGAGAAAGGGAGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCCTCACCACAGC CCTG 47 Donor GFP donor toATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCC DNA AAVS1 locusCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGTAA 48 DonorAAVS1 locus ACTGTGGGGTGGAGGGGACAGATAAAAGTACCCAGAACCAG DNA RHA (used forAGCCACATTAACCGGCCCTGGGAATATAAGGTGGTCCCAGCT GFP donor)CGGGGACACAGGATCCCTGGAGGCAGCAAACATGCTGTCCTGAAGTGGACATAGGGGCCCGGGTTGGAGGAAGAAGACTAGCTGAGCTCTCGGACCCCTGGAAGATGCCATGACAGGGGGCTGGAAGAGCTAGCACAGACTAGAGAGGTAAGGGGGGTAGGGGAGCTGCCCAAATGAAAGGAGTGAGAGGTGACCCGAATCCACAGGAGAACGGGGTGTCCAGGCAAAGAAAGCAAGAGGATGGAGAGGTGGCTAAAGCCAGGGAGACGGGGTACTTTGGGGTTGTCCAGAAAAACGGTGATGATGCAGGCCTACAAGAAGGGGAGGCGGGACGCAAGGGAGACATCCGTCGGAGAAGGCCATCCTAAGAAACGAGAGATGGCACAGGCCCCAGAAGGAGAAGGAAAAGGGAACCCAGCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGCCGAGAAGGAAGTGCTCCGGAAAGAGCATCCTTGGGCAGCAACACAGCAGAGAGCAAGGGGAAGAGGGAGTGGAGGAAGACGGAACCTGAAGGAGGCGGCAGGGAAGGATCTGGGCCAGCCGTAGAGGTGACCCAGGCCACAAGCTGCAGACAGAAAGCGGCACAGGCCCAGGGGAGAGAATGCAGGTCAGAGAAAGCAGGACCTGCCTGGGAAGGGGAAACAGTGGGCCAGAGGCGGCGCAGAAGCCAGTAGAGCTCAAAGTGGTCCGGACTCAGGAGAGAGACGGCAGCGTTAGAGGGCAGAGTTCCGGCGGCACAGCAAGGGCACTCGGGGGCGAGAGGAGGGCAGCGCAAAGTGACAATGGCCAGGGCCAGGCAGATAGA CCAGACTGAGCTATGG 49 DonorHBB locus CTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA DNA LHA (used forCCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC E7 to E7VAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACT AAV.304)TTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCA 50 Donor E7 to E7VTCTGACTCCTGTCGAGAAGTCTGCAGTCACTGCTCTATGGGG DNA AAV.304 GAAA 51 DonorHBB locus GTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTG DNA RHA (used forGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAA E7 to E7VCTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAG AAV.304)GCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGTC 52 Donor HBB locusCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA DNA LHA (used forCCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC E7 to E7VAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACT AAV.307)TTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCA 53 Donor E7 to E7VTCTGACTCCTGTCGAAAAATCCGCTGTCACCGCCCTCTGGGG DNA AAV.307 CAAG 54 DonorHBB locus GTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTG DNA RHA (used forGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAA E7 to E7VCTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAG AAV.307)GCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGTC 55 Donor HBB locusGACTGCATTAAGAGGTCTCTAGTTTTTTACCTCTTGTTTCCCA DNA LHA (used forAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTA GFP AAV)TTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTACATATACACATATATATATATTTTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACTGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAATTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGAGGA 56 Donor GFPATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCC DNACATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGTAA 57 DonorHBB locus CTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTG DNA RHA (used forAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTA GFP AAV)AGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAA TTTTGCATTTGTAATTTTAAAAAATGC58 MND GGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTATGG promoterGGATCCGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAG 59 EF1αGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACA promoterGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTG A 60 SFFVGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAG promoterAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCG CGCCAGTCCTCCGACAGACTGAGTCG 612A peptide GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA from porcineAGAAAACCCCGGTCCT teschovirus 62 SyntheticAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTT poly(A) signal TTGTGTG 63 PAMCanonical N_(x)NRG (N = any nucleotide; R = A or G; x = 19-21) PAM 64PAM SpCas9 PAM NRG (N = any nucleotide, R = A or G) 65 NuclearSV40 NLS 1 PKKKRKV localization signal (NLS) 66 NLS SV40 NLS 2 PKKKRRV67 NLS Nucleoplasmin KRPAATKKAGQAKKKK NLS 68 I53 i53 (DNA)ATGCTGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACCCTGGAGGTGGAGCCCAGCGACACCATCGAGAACGTGAAGGCCAAGATCCAGGACAAGGAGGGCATCCCCCCCGACCAGCAGAGGCTGGCCTTCGCCGGCAAGAGCCTGGAGGACGGCAGGACCCTGAGCGACTACAACATCCTGAAGGACAGCAAGCTGCACCC CCTGCTGAGGCTGAGGTGA 69 I53i53 (RNA) AUGCUGAUCUUCGUGAAGACCCUGACCGGCAAGACCAUCA mRNACCCUGGAGGUGGAGCCCAGCGACACCAUCGAGAACGUGAAGGCCAAGAUCCAGGACAAGGAGGGCAUCCCCCCCGACCAGCAGAGGCUGGCCUUCGCCGGCAAGAGCCUGGAGGACGGCAGGACCCUGAGCGACUACAACAUCCUGAAGGACAGCAAGCUG CACCCCCUGCUGAGGCUGAGGUGA 70 I53i53 (aa) MLIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLAFAGKSLEDGRTLSDYNILKDSKLHPLLRLR 71 DM I53-DMAUGUUGAUUUUCGUGAAAACCCUUACCGGGAAAACCAUCA mRNA (RNA)CCCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGGCCUUUGCUGGCAAAUCGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCUAAAGGACUCUAAACU UCAUCUAGUGUUGAGACUUCGU 72A10 (DNA) ATGCAGATTTACGTGAAGACCTTTGCCCGGAAGCCCATCACCCTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGCGACTGATCTTTGCTGAAATGCGGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTAAAAACGACTCTACTCTTTTTCTTGT GTTGAAAAATAGTGTTACT 73A10 (RNA) AUGCAGAUUUACGUGAAGACCUUUGCCCGGAAGCCCAUCACCCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGCGACUGAUCUUUGCUGAAAUGCGGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUAAAAACGACUCUACUCU UUUUCUUGUGUUGAAAAAUAGUGUUACU 74A10 (aa) MQIYVKTFARKPITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAEMRLEDGRTLSDYNIKNDSTLFLVLKNSVT 75 A11 (DNA)ATGCTGATTTTCGTGACCACCGATATGGGGATGACAATCTCACTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGATCTTTGGTGACAAGGATCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCAAAAGGAGTCTAGCCTTAATCTTG TGCTGAAACTTCGTGGTGGT 76A11 (RNA) AUGCUGAUUUUCGUGACCACCGAUAUGGGGAUGACAAUCUCACUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGAUCUUUGGUGACAAGGAUCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUAGCCU UAAUCUUGUGCUGAAACUUCGUGGUGGU 77A11 (aa) MLIFVTTDMGMTISLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFGDKDLEDGRTLSDYNIQKESSLNLVLKLRGG 78 C08 (DNA)ATGCAGATTTTCGTGACCACCGATATGTGGATGAGAATCTCACTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGATCTTTGGTGACAAGGATCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCAAAAGGAGTCTAGCCTTAATCTTG TGCTGAACCTTCGTGGTGGT 79C08 (RNA) AUGCAGAUUUUCGUGACCACCGAUAUGUGGAUGAGAAUCUCACUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGAUCUUUGGUGACAAGGAUCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUAGCCU UAAUCUUGUGCUGAACCUUCGUGGUGGU 80C08 (aa) MQIFVTTDMWMRISLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFGDKDLEDGRTLSDYNIQKESSLNLVLNLRGG 81 G08 (DNA)ATGTTGATTTTCGTGAAAACCCTTACCGGGAAAACCATCACCCTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGATCTTTGCTGGCAAATCGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCTAAAGGACTCTAAACTTCATCCTCT GTTGAGACTTCGTGGTGGT 82G08 (RNA) AUGUUGAUUUUCGUGAAAACCCUUACCGGGAAAACCAUCACCCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGAUCUUUGCUGGCAAAUCGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCUAAAGGACUCUAAACU UCAUCCUCUGUUGAGACUUCGUGGUGGU 83G08 (aa) MLIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKSLEDGRTLSDYNILKDSKLHPLLRLRGG 84 H04 (DNA)ATGCGAATTATCGTGAAAACCTTTATGCGGAAGCCGATCACGCTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGTATTTTGCGGCCAGTCAGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCAAAAGGAGTCTACTCTTCTTCTTGT GGTAAGGCTGCTCCGCGTT 85H04 (RNA) AUGCGAAUUAUCGUGAAAACCUUUAUGCGGAAGCCGAUCACGCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGUAUUUUGCGGCCAGUCAGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUACUCU UCUUCUUGUGGUAAGGCUGCUCCGCGUU 86H04 (aa) MRIIVKTFMRKPITLEVEPSDTIENVKAKIQDKEGIPPDQQRLYFAASQLEDGRTLSDYNIQKESTLLLVVRLLRV 87 I53 alt (DNA)ATGTTGATTTTCGTGAAAACCCTTACCGGGAAAACCATCACCCTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGGCCTTTGCTGGCAAATCGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCTAAAGGACTCTAAACTTCATCCTCT GTTGAGACTTCGT 88I53 alt (RNA) AUGUUGAUUUUCGUGAAAACCCUUACCGGGAAAACCAUCACCCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGGCCUUUGCUGGCAAAUCGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCUAAAGGACUCUAAACU UCAUCCUCUGUUGAGACUUCGU 89FLAG-tagged ATGGACTACAAAGACGATGACGATAAAGCCGCCAGTTTAAA i53 DNACGGCGCGCCATTAATTAAGGATCCAATGTTGATTTTCGTGAAAACCCTTACCGGGAAAACCATCACCCTCGAGGTTGAACCCTCGGATACGATAGAAAATGTAAAGGCCAAGATCCAGGATAAGGAAGGAATTCCTCCTGATCAGCAGAGACTGGCCTTTGCTGGCAAATCGCTGGAAGATGGACGTACTTTGTCTGACTACAATATTCTAAAGGACTCTAAACTTCATCCTCTGTTGAGACTTCGTTGA 90 FLAG-taggedAUGGACUACAAAGACGAUGACGAUAAAGCCGCCAGUUUAA i53 RNAACGGCGCGCCAUUAAUUAAGGAUCCAAUGUUGAUUUUCGUGAAAACCCUUACCGGGAAAACCAUCACCCUCGAGGUUGAACCCUCGGAUACGAUAGAAAAUGUAAAGGCCAAGAUCCAGGAUAAGGAAGGAAUUCCUCCUGAUCAGCAGAGACUGGCCUUUGCUGGCAAAUCGCUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCUAAAGGACUCUAAACUUCAUCCUCUGUUGA GACUUCGUUGA 91 Linker (DNA)GCCGCCAGTTTAAACGGCGCGCCATTAATTAAGGATCCA 92 Linker (RNA)GCCGCCAGUUUAAACGGCGCGCCAUUAAUUAAGGAUCCA 93 Linker AASLNGAPLIKDP 94Protein 6xHis HHHHHH tag 95 Protein Flag MDYKDDDDK tag 96 ProteinFLAG (DNA) GACTACAAAGACGATGACGATAAA tag 97 Protein FLAG (RNA)GACUACAAAGACGAUGACGAUAAA tag 98 Donor HBB locusCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA DNA LHA to RHACCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC (used for E6VAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACT to E6,TTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAAC AAV.323)AGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGAAGAAAAATCCGCTGTCACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGT C 99 Donor HBB locusCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA DNA LHA (used forCCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC E6V to E6,AGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACT AAV.323)TTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTG CATCTGACTCCT 100 DonorHBB locus ACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGG DNA RHA (used forTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTT E6V to E6,TAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAG AAV.323)ACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTA TTACCCTGTC 101 DonorHBB exons 1- ATGGTGCATCTGACTCCTGAAGAAAAATCCGCTGTCACTGCC DNA 3CTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAA TGCCCTGGCCCACAAGTATCAC 102Donor E6V to E6 GAAGAAAAATCCGCTGTC DNA Insert (reverse complement of PAMunderlined) 103 Wild-type S. MAPKKKRKVGSGGSGGSGDKKYSIGLDIGTNSVGWAVITDEYKpyogenes VPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR Cas9RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSAGSGGSGGSGPKKKRKV 104 Wild-type S.MHHHHHHHHGSGGSGGSGPKKKRKVGSGGSGGSGKRNYILGL aureus Cas9DIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGGSAGSGGSGGSGPKK KRKV 105 Donor Full AAV-323CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC templateGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAACCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCCAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGAGAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAAGAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGAAGAAAAATCCGCTGTCACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGTCGGTAACCACGTGCGGCCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 106 AAV2 ITR forCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC LHAGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG (AAV.323)CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGG TTCCT 107 AAV2 ITR forAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCG RHACTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC (AAV.323)GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCG CGCAGCTGCCTGCAGG 108 DonorHBB Locus CTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA templateLHA to RHA CCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC (AAV.304)AGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGTCGAGAAGTCTGCAGTCACTGCTCTATGGGGGAAAGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGT C 109 Donor Full AAVCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC template (AAV.304)GGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAACCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCCAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGTCGAGAAGTCTGCAGTCACTGCTCTATGGGGGAAAGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGTCGGTAACCACGTGCGGCCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 110 Donor HBB LocusCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAA template LHA to RHACCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCC (AAV.307)AGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGTCGAAAAATCCGCTGTCACCGCCCTCTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGT C ill Donor Full AAVCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC template (AAV.307)GGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAACCTATATTATAATTTCATAAAGTCTGTGCATTTTCTTTGACCCAGGATATTTGCAAAAGACATATTCAAACTTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGGTCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAAATCCAAAATCTAACAGCCAAGTCAAATCTGCATGTTTTAACATTTAAAATATTTTAAAGACGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGATCCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTATCCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCATAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAGTACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAATAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTGATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATTGTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCTGAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCGACATGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTGAGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAATAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGTACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGAAAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAACTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATAATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCTTTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAGTTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGTGACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTGTTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATTTTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGCATATATATGTATATGTATGTGTGTATATATACACACATATATATATATATTTTTTCTTTTCTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACCGAGGTAGAGTTTTCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCTACATATCCCAAAGCTGAATTATGGTAGACAAAACTCTTCCACTTTTAGTGCATCAACTTCTTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATTCCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGCATCTGACTCCTGTCGAAAAATCCGCTGTCACCGCCCTCTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGTGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCATATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGTCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACTATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAGAAGAATTTGAAAGGCGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTGTCGGTAACCACGTGCGGCCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 112 Donor 5′ ITRCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCC templateGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCT

1. A method for homology directed repair (HDR) of a double-strand break(DSB) in a target region in a human beta-globin (HBB) gene in a cell orpopulation of cells the method comprising contacting the cell orpopulation of cells with: (a) a S. pyogenes Cas9 endonuclease, an mRNAencoding the S. pyogenes Cas9 endonuclease, or a recombinant expressionvector comprising a nucleotide sequence encoding the S. pyogenes Cas9endonuclease, wherein the S. pyogenes Cas9 endonuclease is a highfidelity Cas9; (b) a single guide RNA (sgRNA) targeting a target site inan HBB gene, the sgRNA comprising a spacer sequence corresponding to atarget sequence consisting of SEQ ID NO: 15; and (c) a recombinantvector comprising a nucleic acid, the nucleic acid comprising from 5′ to3′ (i) a nucleotide sequence homologous with a region located upstreamof the target region in the HBB gene, (ii) a nucleotide sequencehomologous with a region of the HBB gene comprising the target region,the nucleotide sequence comprising the sequence of SEQ ID NO: 102, and(iii) a nucleotide sequence homologous with a region located downstreamof the target region in the HBB gene; wherein a double-strand break(DSB) occurs at the target site in the HBB gene and the nucleic acid isexchanged with a homologous nucleotide sequence of the HBB gene.
 2. Themethod of claim 1, wherein cleavage of one or more predicted off-targetsites in the cell or population of cells is reduced relative to a cellor population of cells contacted with a wild-type S. pyogenes Cas9.3.-5. (canceled)
 6. The method of claim 1, wherein the nucleotidesequence of (c)(i) is homologous with a region located upstream of theE6V mutation in the HBB gene and the nucleotide sequence of (c)(iii) ishomologous with a region located downstream of the E6V mutation. 7.-12.(canceled)
 13. A method for correcting an E6V mutation in humanbeta-globin (HBB) in a cell or population of cells, the methodcomprising contacting the cell or population of cells comprising an HBBgene encoding the E6V mutation with: (a) a S. pyogenes Cas9endonuclease, an mRNA encoding the S. pyogenes Cas9 endonuclease, or arecombinant expression vector comprising a nucleotide sequence encodingthe S. pyogenes Cas9 endonuclease; (b) a single guide RNA (sgRNA)targeting a target site in the HBB gene, the sgRNA comprising a spacersequence corresponding to a target sequence consisting of SEQ ID NO: 15;and (c) a recombinant vector comprising a nucleic acid, the nucleic acidcomprising from 5′ to 3′ (i) a nucleotide sequence homologous with aregion located upstream of the E6V mutation in the HBB gene, (ii) anucleotide sequence which corrects the E6V mutation and is homologouswith a region of the HBB gene encoding the E6V mutation, the nucleotidesequence set forth in SEQ ID NO: 102, and (iii) a nucleotide sequencehomologous with a region located downstream of the E6V mutation in theHBB gene, wherein a double-strand break (DSB) occurs at the target sitein the HBB gene and the nucleic acid is exchanged with a homologousnucleotide sequence of the HBB gene, thereby correcting the E6V mutationin the HBB gene in the cell or population of cells.
 14. The method ofclaim 1, further comprising contacting the cell with a 53BP1 inhibitor;an inhibitor of DNA-PK; or both. 15.-16. (canceled)
 17. The method ofclaim 14, wherein the 53BP1 inhibitor and/or the inhibitor of DNA-PKincreases HDR of the DSB, relative to HDR in a cell or population ofcells without the 53BP1 inhibitor and/or inhibitor of DNA-PK. 18.-56.(canceled)
 57. The method of claim 1, wherein the high fidelity Cas9endonuclease comprises a R691A mutation.
 58. The method of claim 1,wherein the high fidelity Cas9 endonuclease comprises at least one NLS.59. (canceled)
 60. The method of claim 1, wherein (i) the 53BP1inhibitor and/or the inhibitor of DNA-PK increases HDR frequency in thecell population by at least 50% relative to a cell population withoutthe 53BP1 inhibitor and/or the inhibitor of DNA-PK; (ii) the 53BP1inhibitor and/or the inhibitor of DNA-PK decreases indel frequency by2-10 fold in the cell population; or (iii) both (i) and (ii). 61.(canceled)
 62. The method of claim 14, wherein the 53BP1 inhibitor is a53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB inthe cell, wherein the 53BP1 binding polypeptide comprises an amino acidsequence selected from a group consisting of: SEQ ID NOs: 70, 74, 77,80, 83 and
 86. 63. (canceled)
 64. The method of claim 14, wherein (i)the 53BP1 inhibitor comprises a nucleic acid or a vector comprising anucleotide sequence encoding a 53BP1 binding polypeptide that inhibits53BP1 recruitment to the DSB site in the cell; or (ii) the 53BP1inhibitor comprises a vector comprising a nucleotide sequence encodingthe 53BP1 binding polypeptide, wherein the nucleotide sequence isselected from a group consisting of: SEQ ID NOs: 69, 73, 76, 79, 82, 85and
 88. 65.-68. (canceled)
 69. The method of claim 14, wherein theinhibitor of DNA-PK targets the catalytic subunit of DNA-PK (DNA-PKcs).70. The method of claim 14, wherein the inhibitor of DNA-PK is Nu7441,Compound 984, or Compound
 296. 71. (canceled)
 72. The method of claim 1,wherein (i) tithe nucleotide sequence of (c)(i) is about 0.2 kb to about3 kb in length; (ii) the nucleotide sequence of (c)(iii) is about 0.2 kbto about 3 kb in length; or (iii) both (i) and (ii). 73.-74. (canceled)75. The method of claim 1, wherein the nucleotide sequence of (c)(i)and/or the nucleotide sequence of (c)(iii) is about 2.2 kb each.
 76. Themethod of claim 1, wherein the recombinant vector comprises SEQ ID NO:98.
 77. The method of claim 1, wherein the recombinant vector is an AAVvector.
 78. The method of claim 77, wherein the AAV vector is about 2.5kb-4.6 kb in length.
 79. The method of claim 77, wherein the AAV vectorcomprises AAV6.
 80. The method of claim 77, wherein the AAV vectorcomprises 5′ and 3′ inverted terminal repeats (ITRs) derived from AAV2.81. (canceled)
 82. The method of claim 77, wherein the AAV vectorcomprises SEQ ID NO:
 105. 83. The method of claim 1, wherein (i) thecell or population of cells is a hematopoietic stem or progenitor cell(HSPC) or a population of HSPCs; (ii) the cell or population of cells isa CD34 expressing cell or a population of CD34 expressing cells; or(iii) both (i) and (ii). 84.-85. (canceled)
 86. The method of claim 83,wherein the cell or the population of cells is isolated from a tissuesample obtained from a human donor.
 87. The method of claim 86, whereinthe tissue sample is a peripheral blood sample.
 88. The method of claim86, wherein the human donor has a sickle cell disease.
 89. A cell orpopulation of cells generated by the method of claim
 1. 90. A method fortreating a patient having a disease or disorder, comprisingadministering the cell or population of cells of claim 89, therebytreating the disease or disorder.
 91. (canceled)